KR100491222B1 - Recombinant anti-cd4 antibodies for human theraphy - Google Patents

Abstract

The present invention relates to chimeric antibodies specific for human CD4 antigens having substantially non-T-cell consuming activity, DNA-encoded pharmaceutical compositions containing them and their use as therapeutic agents. These chimeric antibodies contain Old World monkey variable sequences and human constant domain sequences, preferably human gamma 4 or mutant forms thereof. These antibodies have desirable therapeutic properties including low antigenicity, reduced (or absent) T cell consuming activity, good affinity for human CD4 and increased stability (in vivo half-life).

Description

RECOMBINANT ANTI-CD4 ANTIBODIES FOR HUMAN THERAPHY}

The present invention relates to specific recombinant antibodies against CD4 and methods of making such antibodies useful for the treatment of humans.

CD4 is a surface glycoprotein expressed primarily on cells of the T lymphocyte lineage, including most thymic cells and some peripheral T cells. Although low levels of CD4 are also expressed in some nonlymphoid cells, the significance of the function of this diffuse cell distribution is not known. On mature T cells, CD4 has cocognitive functions through interaction with MHC class molecules expressed in antigen presenting cells. CD4 ⁺ T cells primarily constitute a subset of helpers that regulate T and B cell function in T-dependent responses to viral, bacterial, homogeneous and parasitic infections.

During the development of autoimmune diseases, particularly when resistance to autologous antigens is destroyed, CD4 ⁺ T cells contribute to the inflammatory response that leads to joint and tissue destruction. This action is facilitated by the growth of inflammatory cells of the hematopoietic line, the production of antibodies, inflammatory cytokines and mediators, and activation of killer cells.

Rheumatoid arthritis (RA), or the inflammatory disease of the synovial membrane, is a manifestation of autoimmune phenomena leading to soreness, deformation and destruction of the joints. As with most autoimmune diseases, the etiology of RA is not fully understood. However, RA is characterized by an increase in the level of activated CD4 ⁺ T lymphocytes in the joints involved. In general, there is no cure for RA. First line treatment for RA is designed to alleviate RA symptoms and increase ability to act over a short period of time. Second and third line immunosuppressants and steroids, such as azathioprine, methotrexate and prednisolone, which target the underlying disease, are administered to more severe patients, showing only slightly effective or unacceptable toxicity for chronic treatment. In addition, the material does not prevent joint destruction.

In addition to RA, CD4 ⁺ cells are also involved in other chronic diseases including psoriasis, insulin dependent diabetes mellitus, systemic lupus erythematosus and inflammatory bowel disease. CD4 expression may also be related to other autoimmune diseases.

If T cells were involved in the development and maintenance of autoimmune diseases, immunosuppression would have been an important treatment. Available immunosuppressive agents, such as cyclosporin A, have been successfully used to treat transplant rejection. However, their side effects have made them unacceptable for the chronic treatment of autoimmune diseases.

Depletion of the entire T cell population comprising CD4 ⁺ subsets in a clinical setting is achieved by methods including chest tube drainage, total lymphocyte irradiation and lymphocyte production, which induce clinical improvement in some patients. . However, current methods focus on more selective agents that block unwanted immune responses without causing solid organ toxicity or other major side effects. This method that can be achieved is by selective elimination or inactivation of the disease by mediating T cells with monoclonal antibodies (mAbs). MAbs for CD4 represent one of these methods. CD4 mAbs can stop or reverse disease progression when they are administered prophylactically or therapeutically. In addition, early results from some clinical trials on anti-CD4 in RA, psoriasis, inflammatory bowel disease, and systemic vasculitis provide some preliminary evidence of potential therapeutic effects.

In fact, the purpose of anti-CD4 mAbs treatment is to stop the suicide activity of CD4 ⁺ cells, especially during the acute phase of autoimmune disease. The final therapeutic goal is to impose long-term resistance to the state of immunological non-response (anergy) or seizure antigens (or specific tissues) to maintain the underlying disease without compromising normal host defenses against opportunistic infections. will be. In addition to RA, CD4 mAbs may also be beneficial for the treatment of other autoimmune diseases such as insulin dependent diabetes mellitus, systemic lupus erythematosus, psoriasis, inflammatory bowel disease and multiple sclerosis.

Because of the potential importance of anti-CD4 mAbs as an immunotherapeutic agent, anti-CD4 mAbs as potential therapeutic agents have been studied in numerous companies and research institutions. For example, Centercor reported an anti-CD4 mAb referred to as Centra, a chimeric murine mAb for CD4. In addition, Johnson & Johson / Ortho reported OKT-4a, an anti-CD4 mAb, a humanized murine mAb. Burroughs Wellcome also reported the anti-CD4 mAb, a humanized murine mAb for CD4. In addition, Sandoz and MedImmune (in collaboration with Merck) have developed anti-CD4 murine-humanized mAbs specific for CD4. In addition, Becton Dickinson, Immunotech and Boehringer Mannheim all developed anti-CD4 mAbs.

In addition to anti-CD4 mAbs, various immunomodulators and agents have been reported to have potential applicability for RA treatment. Such immunomodulators and medicaments include, for example, cell adhesion blockers, cytokine receptor blockers, immunotoxins and T cell receptor antagonists. Specific examples include gamma interferon, anti-CAM-1 (leukocyte transport, murine anti-CD54 mAb to block adhesion), Campas-1H (rat-humanized anti-CDw52 mAb), IL-1 receptor, cA2 (TNF-alpha chimeric mAb), CDP 571 (anti-TNF mAb), anti-IL-2R (humanized-murine anti-CD25 mAb), SDZ CHH 380 (murine-human anti-CD7 mAb), DAB486 IL-2 (Nonspecific to IL-2 fusion toxins, CD4 and CD8 cells), Antril (IL-1RA), anti-TCR (mAbs and proteins targeting T cell receptor subsets) and ComaZyme- CD5 (murine anti-CD5 toxin conjugate).

In addition, other immunomodulators and immunosuppressants with potential applicability to the treatment of autoimmune diseases include rapamycin (oral immunosuppressants), therafectin, Leflunomide (immunosuppressor prodrugs), teni Tenidap (cytokine modulators / CO-inhibitors), IMM-125 and RS-61443 (oral immunosuppressants).

As mentioned above, a number of monoclonal antibodies against CD4 have been reported with potential therapeutic applicability. For the most part, such antibodies include murine mAbs, chimeric or murine-humanized antibody-CD4 mAbs.

Murine monoclonal antibodies are diagnostic and clinical trials as treatments for the treatment of both acute and chronic human diseases including leukemia, lymphoma, solid tumors (e.g., colon, chest, hepatic tumors), AIDS and autoimmune diseases. Has potential utility in. However, murine antibodies are disadvantageous because they often induce an immune antibody response in the host against murine monoclonal antibodies.

Mouse / human chimeric antibodies have also been reported. The antibody includes the binding characteristics and effector function of parental mouse antibodies associated with human constant regions. Cabilly et al., US Patent No. 4,816,567; Shoemaker et al., US Patent No. 4, 978,775; Beavers et al., US Patent No. 4,975,369; And Boss et al., US Patent No. 4,816,397 are all incorporated herein by reference. Typically, the chimeric antibody consists of a genome gene library constructed from DNA extracted from existing murine hybridomas (Nishman et al., 47 Cancer Research , 999 (1987)). The library was then screened for the variable region genes of both the heavy and light chains showing the correct antibody fragment rearrangement pattern. The cloned variable region genes are then ligated into expression vectors containing cloned cassettes of sufficient heavy or light chain human constant region genes. The chimeric gene is then expressed in the selected cell line, typically the murine myeloma lineage.

However, even if such chimeric antibodies are used to treat humans, they also have some problems. Similar to murine monoclonal antibodies, human recipients can produce antibodies against chimeric antibodies. This is detrimental to the efficacy of continued treatment with chimeric antibodies.

In improvements to conventional chimeric antibodies, some researchers have found a method for making human monoclonal antibodies that do not cause this problem. See, for example, Erlich et al., 34 Clinical Chemistry, 1681 (1988); Erlich et al., 7 Hybridoma, 385 (1998); Erlich et al., 6 Hybridoma, 151 (1987) and Erlich et al., 1 Human Antibody Hybridomas, 23 (1990)]. The reference also hypothesizes that nonhuman primate antibodies, such as chimpanzee monoclonal antibodies, have good resistance in humans due to structural similarities with human antibodies. However, antibody production in humans is morally contraindicated.

Because human antibodies are non-immunogenic in rhesus monkeys (ie, do not cause antibody responses), Ellich et al. Also predicted that primate antibodies would be non-immunogenic in humans. Elk et al. (Id.) Disclose that if a primate antibody has the same constant region as that of a human immunoglobulin, or at least the structure of the antibody differs from the human immunoglobulin in a degree to which the antibody structures of different humans differ from one another, It is pointed out that testing is unnecessary. Thus, they suggest that chimpanzee antibodies may be useful for treating humans.

Improvements to known chimeric antibodies that are often antigenic in humans are described in US Application Nos. 08 / 476,237 filed June 7, 1995, 08 / 347,072, filed January 25, 1995. Application), 07 / 912,212 (filed July 10, 1992), 07 / 856,281 (filed March 23, 1992) and 07 / 735,064 (filed July 25, 1991) And the preparation of Old World monkey (eg, baboon or macaque) monoclonal antibodies and chimeric antibodies derived therefrom are generated by recombinant methods containing the variable domains of Old World monkey antibodies, and cloned Methods for fusing to humans, chimpanzees or other monkey constant regions or other monkey framework regions are described. In particular, the application describes the use of such chimeric recombinant antibodies as immunotherapeutic agents for the production of the old world monkey antibodies against human antigens and chimeric antibodies derived therefrom and for the treatment of human diseases.

The applications differ from chimpanzees in that evolutionarily distanced monkeys (eg, baboon or macaque monkeys (including crab monkeys and rhesus monkeys)), such as, for example, CD4 and CD54 in these monkeys. Even for relatively conserved human antigens, these monkey antibodies or recombinant chimeric antibodies derived therefrom are sufficiently different from humans to allow antibodies to human antigens and similar enough to have structurally similar antibodies to human antibodies. Is based on the surprising finding that no host anti-antibody reactions occur when introduced into humans.

Unlike some of the conventional antibodies used in the treatment of humans, including the known chimeric antibodies in the above applications, such chimeric antibodies have several disadvantages, for example 1) human anti-antibodies to repeated administrations necessary to treat chronic diseases. Immunogenicity and induction of (HAA) responses, 2) relatively short half-life compared to human antibodies, and 3) lack of effector action of human cells or complementation.

The lack of this disadvantage is very advantageous in the treatment of humans. For example, for chronic human disease, including autoimmune diseases, or another disease requiring long-term administration of the antibody, one of the major obstacles to repeated antibody treatment is the host response to the therapeutic antibody. HAA responses are often unpredictable in one patient and another. In addition, such a response is not exclusively but distinctly induced against the constant region of the antibody molecule, which interferes or reduces the therapeutic effect with the antibody or another antibody of the same isotype. The recombinant chimeric antibodies described in the above-mentioned applications can avoid this problem, allow the production of antibodies of suitable specificity and desired effector function, and use such antibodies in the production of recombinant antibodies.

Such recombinant antibodies include variable regions of antibodies derived from immunized monkeys required for antigen binding and suitable portions of constant regions of antibodies from humans or chimpanzees. Thus, they enable the maintenance of the specificity and high affinity of the monkey monoclonal antibodies and the desired effector function by suitable selection of human or chimpanzee constant regions.

Some of these related applications illustrate monkey / human chimeric antibodies with specificity for CD4, in particular referred to as CE9.1, which antibodies are anti-CD4 monoclonal generated in cynomolgus monkey and human immunoglobulin light chain lambda constant regions. Heavy and light chain variable domains of the null antibody, and human immunoglobulin heavy chain gamma 1 constant region. The antibody has some T cell clearance activity but lower activity compared to previous CD4 monoclonal antibodies. However, it is desirable to prepare antibodies with lower activity or no T cell scavenging activity, as this may improve the therapeutic potential of the antibody.

The applications also describe preferred vector systems for the preparation of such chimeric antibodies, in particular TCAE 5.2 and TCAE 6 comprising the following features:

1) 4 transfer cassettes in tandem:

(a) Human immunoglobulin light chain constant regions: in TCAE 5.2, these are human immunoglobulin kappa light chain constant regions (Kabat number amino acids 108-214, allotype Km 3) and in TCAE 6 human immunoglobulin light chain lambda constants Region (Kabat number amino acids 108-215, genotype Oz minus, Mcg minus, Ke minus allotype).

(b) human immunoglobulin heavy chain constant regions; In both constructs the human immunoglobulin heavy chain is a gamma / constant region (Kabat number amino acids 114-478 allotype Gm1a, Gm 12).

(c) DHFR; Containing their own eukaryotic promoter and polyadenylation region; And

(d) NEO; It also contains their own eukaryotic promoter and polyadenylation region.

2) human immunoglobulin light and heavy chain cassettes contain synthetic signal sequences for the secretion of immunoglobulin chains;

3) Human immunoglobulin light and heavy chain cassettes contain specific DNA, which allows insertion of light and heavy chain immunoglobulin variable regions, maintains a translation reading frame, and incorporates amino acids commonly found in immunoglobulin chains. Do not change.

However, despite the above, there is still a need in the art for improved antibodies specific for CD4 and with low antigenicity in humans, which are for example in the treatment of autoimmune diseases such as rheumatoid arthritis. It can be used therapeutically. In particular, there is a need to prepare anti-CD4 antibodies with improved properties, eg, longer half-life and / or substantially lacking or lacking clearance activity.

Object of the present invention

In this part, the object of the present invention is a novel monoclonal specific for CD4, with improved properties, for example longer half-life, lower immunogenicity in humans and / or reduced or absent T cell clearance activity. It is to provide a local chimeric antibody. In particular, it is an object of the invention to be specific for CD4, and human or monkey constant domain sequences, in particular human kappa or lambda light chain constant regions, and human gamma 1, having modified effector function and improved stability over gamma 4 isotypes. To prepare an anti-CD4 chimeric antibody containing the antigen recognition portion of Old World monkey immunoglobulin specific for gamma 4 or modified gamma 4 human heavy chain constant region sequences.

A more specific object of the invention is a modified effector function and improved compared to monkey and human constant domain sequences, preferably human kappa or lambda light chain constant domain sequences and human gamma 1 or gamma 4 constant domain sequences or gamma 4 isotypes. Novel monoclonal chimeric antibody containing the specific monkey anti-CD4 variable heavy chain sequence shown in FIG. 1 and the monkey anti-CD4 variable light chain sequence shown in FIG. 2 fused to a human mutant gamma 4 heavy chain with stability. Is in providing.

It is another object of the present invention to provide such improved chimeric anti-CD4 antibodies and vectors, and vectors and host cells that can be used for expression of the chimeric anti-CD4 antibodies. Preferably such vectors will include the expression vectors referenced in the applications cited herein by reference, with the host cell being preferably a CHO cell.

Another object of the present invention is to provide a pharmaceutical composition for use in the treatment or prevention of a disease associated with CD4, in particular an autoimmune disease, the composition comprising a prophylactic or therapeutically effective amount in combination with a pharmaceutically acceptable carrier. Contains an improved chimeric anti-CD4 antibody of the invention.

Another object of the present invention is to provide a method for the treatment or prophylaxis of CD4-related diseases, in particular autoimmune diseases and other diseases, wherein immunosuppression is carried out in a prophylactically or therapeutically effective amount with a pharmaceutically acceptable carrier. By administration of the novel chimeric anti-CD4 antibody of the invention.

Brief description of the drawings

1 depicts the amino acid and DNA sequences of the heavy chain variable domains of CE9.1.

Figure 2 depicts the amino acid and DNA sequences of the light chain variable domains of CE9.1.

Figure 3 depicts amino acid sequences and DNA sequences of human lambda variable and constant domains in CE9.1.

4 depicts DNA and amino acid sequences encoding heavy chain variable and constant gamma 4 sequences.

Figure 5 depicts DNA and amino acid sequences encoding human heavy chain gamma 4 containing E mutations.

Figure 6 depicts DNA and amino acid sequences encoding human heavy chain gamma 4 containing P and E mutations.

7-1, 7-2 and 8 illustrate nucleic acid sequences of various leader sequences useful in the present invention.

9 shows a scattergram in which CE9.1 binds to fresh human PMNC, where panel A of the quadrant represents lymphocytes dually stained with CE9.1 and OKT3, and panel B is CE9 Populations stained double with .1 and OKT4 are shown, panel C shows the absence of cells stained double with CD8 and CE9.1, and panel D control shows cells stained with normal and human IgG.

10A, 10B and 10C show the Fc receptor binding properties of CE9.1, where measurements are a) IFN induced fresh monocytes (here, F (ab ′) 2 fragments of CE9.1 as negative controls) Used), b) flow cytometric histogram of CD4 ⁺ aggregation of binding fibroblasts with IFN induced or uninduced fresh monocytes and c) INF induced fresh monocytes in the presence or absence of antibody It is shown.

FIG. 11 depicts inhibition of human mixed lymphocyte response by CE9.1, where a) fresh human PBL is used as a reactant and mitocin C-treated stimulator cells from unrelated donors Used to test concentration range inhibition properties, inhibition is measured by the amount of thymidine incorporation of the IL-2 product, and b) using chimpanzee reactants and unrelated chimpanzee stimulants (Leu3a, murine anti-human CD4) MLR was used as a control.

Figure 12 depicts cytotoxicity of antibody dependent cells of CE9.1, where lysis of SupT-18 target cells in the presence of interferon stimulates effector cells and 4D9 is a murine anti-CD4 monoclonal antibody IgG2a.

FIG. 13 shows flow cytometry histogram of C1q binding to SupT1-18 cells in the presence and absence of CE9.1, where 10,000 events were recorded, results are presented as bar graphs, and PR0945 is a high anti-CD4 serum Polyclonal antibodies from monkeys with appropriate concentrations and negative controls are C1q and anti-C1q in the presence of CE9.1.

Figure 14 shows a complement dependent cytotoxicity assay of CE9.1, where cytotoxicity is lysis of SupT-18 cells in the presence of CE9.1 and rabbit complement, 4D9 is a subclass IgG2a that can be immobilized to complement Is a murine anti-CD4 control, and PR0965 is a polyclonal mixture of antibodies from the sera of cynomolgus monkeys with high anti-CD4 titers.

FIG. 15 depicts a high dose pharmacological study in six chimpanzees, where CD4, CD8 levels in peripheral blood are expressed over a 150-300 day period, and a CD3-CD8 curve showing the number of CD4 regulatory cells is also shown. have; Top panel: Group 1-Number of chimpanzees monitored. Arrows indicate CE9.1 doses. (2) Saline control, middle panel: chimpanzees (CE) receiving CE9.1 of Group 2-10 mg / kg. When the number of CD4 has returned back within 30% of petroleum jelly, administration is repeated. Bottom panel: Chimpanzees (2) awarded CE9.1 of Group 3-10 mg / kg. When the number of CD4 is back within 70% of petroleum jelly, dosing is repeated.

16 depicts PCR primers suitable for obtaining human 4 constant regions.

17 depicts the CE94PE heavy chain sequence.

FIG. 18 depicts non-reducing SDS-polyacrylamide gel electrophoresis of CE9.1, CE94 (G4), CE94E (G4E) and CE9PE (G4PE). Halfmer molecules exhibit a molecular weight of about 80 kD.

19 contains data for association and dissociation of SPR progression curves.

20 depicts the effect of CD4 mAb constructs on primary MLR.

Figure 21 depicts the attachment of IFN-induced monocyte line THP-1 to CD4 + fibroblast transfectants.

FIG. 23 shows CDC and ADCC results using murine fixed mAbs for CE94PE, CE9.1 and HuCD4.

FIG. 24 shows plasma concentrations after administration of 1 mg / kg of CE94E and CE94PE pills in mouse Sprague-Dawley mice.

Figure 25 depicts the effect of treatment with mAb in egg albumin-specific antibody responses in HuCD4 transgenic mice.

Detailed description of the invention

The present invention is an Old World monkey anti-CD4 monoclonal fused to a desired monkey or human constant domain sequence, preferably a gamma 1, gamma 4 or mutated gamma 4 human heavy chain constant domain and human kappa or lambda light chain constant domain sequence. Provided are novel monoclonal chimeric antibodies specific for CD4 containing the antigen binding portion of the variable region of the antibody. Such antibodies exhibit improved properties with respect to conventional anti-CD4 monoclonal antibodies, for example, high affinity for human CD4, with little or no immunogenicity in humans. The gamma 4 version exhibits a decrease or absence of effector function, such as Fc receptor binding activity or complement fixation, with little or no T cell depleting activity.

Methods for obtaining old world monkey monoclonal antibodies specific for CD4 and clones that produce old world monkey monoclonal antibodies specific for CD4 can be found in the related patent applications referenced above, incorporated herein by reference.

Typically, they include Old World monkey immunization against human CD4 under conditions such as Old World monkeys producing anti-CD4 antibodies; And for example, the hybridoma fusion, herpes wave ACF (Herpes papio) using a virus transformed, single B- cell cloning (also "transient immortalization" as referred to) -CD4 and wherein by the generation of a library of recombinant immunoglobulins Immortalization of monkey cells resulting in antibody production. In a preferred embodiment, the method comprises B-cell selection of monkeys from peripheral blood leukocytes, spleen, bone marrow and lymph nodes; Core selection to produce suitable antibodies; Immunoglobulin gene rescue encoding antibodies from immortalized cell lines; And gene expression in producer cell lines (ie, cell lines capable of producing sufficient antibodies useful for human treatment). As defined in the above referenced documents, Old World monkeys include baboons and macaques (including rhesus monkeys and cynomolgus monkeys).

As mentioned above, in a preferred embodiment, the chimeric antibodies of the invention will comprise the anti-CD4 Old World monkey variable heavy chain sequence and variable light chain sequence shown in FIGS. 1 and 2 fused to human constant domain sequences. Suitable methods for obtaining such specific variable heavy and variable light domain sequences are described in US Application 08 / 476,237, filed June 7, 1990, 08 / 397,072 filed on January 25, 1995. 07 / 912,292, filed Jul. 10, 1992, all of which are incorporated herein by reference. The application also lists the entire nucleic acid and amino sequence of the sequence.

Such variable heavy chain domain sequences can be fused to any desired human constant domain sequence. Certain choices will affect the effector function of the resulting chimeric anti-CD4 antibody. Preferably, the human heavy chain constant domain will comprise gamma 1, gamma 4, or modified gamma 4 constant domain referred to herein as gamma 4E or modified gamma 4 referred to herein as 4PE. The selection of gamma 4 is useful because it has been found to produce chimeric antibodies that lack T cells or substantially lack T cell clearance activity (80-100% in proportion to gamma 1). This is believed to be due to the gamma 4 constant domain being unable to bind complement. In addition, the constant domains may be modified to enhance the properties of the resulting chimeric antibody, eg, stability, and / or to remove clearance activity. In particular, the P and E modifications of the gamma 4 domains described below are modifications of gamma 4 in the hinge region that provide enhanced stability and eliminate elimination activity. In addition, other modifications are expected to provide chimeric antibodies with improved properties.

It is preferable that the human light chain constant domain contained in the main chimeric anti-CD4 antibody is a human kappa or lambda light chain constant region, more preferably a human lambda light chain constant region. Amino acid and DNA sequences encoding human gamma 1, gamma 4, kappa and lambda constant domains are also known in the art. In addition, amino acid and nucleic acid sequences, and lambda constant domain sequences for human gamma 4, and E and PE mutations are shown in FIGS. 4-6 and 3, respectively.

Illustrative embodiments of the invention include specific chimeric anti-CD4 monoclonal antibodies referred to as CE9.1 containing antigen binding domains obtained from human sCD4-immunized cynomolgus monkeys with the constant domains of human IgG1 and their Monoclonal chimeric antibodies derived from, eg, CE94, CE94K, CE94E, and CE94PE, wherein the derived monoclonal chimeric antibodies have the same antigen binding domain of CE9.1, but are engineered by genetic engineering Human IgG4 Fc binding domain framework. Monoclonal antibody CE94E contains a mutation (L236E) in which leucine is substituted with glutamic acid near the hinge region of the antibody (E modification). The monoclonal antibody CE94PE contains a mutation in which leucine is substituted with glutamic acid and a mutation in which serine is substituted with proline (S229P) (“E” and “P” modifications). CE94K antibodies differ from CE94 by replacement of their light chain constant regions from human K to human subtypes.

Since constant biological responses of IgG antibodies are known to be dependent on the composition of their carboxy-terminal domains, ie their isotypes, these constant domain switches and mutations are produced. Thus, by modification of the antibody isotype by protein engineering, it may be possible to modify the biological response of IgG antibodies, more particularly major chimeric anti-CD4 monoclonal antibodies.

A desirable outcome of this engineering method is that isotype switching of the Fc portion of the antibody does not reduce the binding affinity of the CD4 antigen that binds to the Fab region. However, this is not known from the outset. Thus, it was possible that changes in the constant regions and their modifications could adversely affect CD4 binding. Thus, the resulting antibodies were analyzed to determine the effect of modification on antibody properties, in particular CD4 antigen binding properties. Known assays can be used to determine the possible effects of constant domain switching on antigen binding. In particular, studies of the interaction between CD4 and CE9.1, CE94, CE94k, CE94E and CE94PE were performed by Scatchard analysis and surface plasmon reaction (SPR). The results of this analysis demonstrated the same CD4 binding to each of the antibodies tested. The mean dissociation constant of CD4 bound to the antibody at 25 was about 1.0nanomolar, revealed by SPR. The measurement also demonstrates the following:

1) CD4 binding to the antibody is 2-site independent and occurs by the same binding model,

2) The functional binding properties of the antigen binding domains are independent of the structural modifications made to the Fc portion of an antibody comprising gamma 1, gamma 4, or modified gamma 4 isotypes. Thus, the present invention may be useful for the method of treating antibodies without isotype switching between IgG1 and IgG4 without loss of antigen binding, particularly antigen binding affinity for CD4.

Furthermore, as mentioned below, replacement of gamma 1 constant domains with gamma 4 substantially reduces Fc receptor binding, complement fixation, and T cell clearance activity, and E and P modifications, respectively, result in Fc receptor binding and T cell clearance. It further removes the activity and provides enhanced antibody activity. Thus, other chimeric antibodies produced in accordance with the invention (antibodies treated with human gamma 4 constant domains or modified forms thereof) are those that substantially lack or completely eliminate T cell clearance activity and / or exhibit improved stability. It is reasonable to assume that the Fc effector function can be selected. Assays for T cell clearance activity, Fc effector function and antibody stability are known in the art.

Accordingly, the present invention provides specific recombinant antibodies that are primate / human chimeric monoclonal antibodies directed against human CD4 antigens that exhibit improved properties, eg, low T cell clearance activity and improved stability. The recombinant antibodies provided with these properties have particular utility as immune-modulators, such as rheumatoid arthritis, psoriasis, systemic lupus erythematosus (SLE), as well as graft-versus-host disease (GVHD), transplant rejection, asthma and HIV. Visa is particularly useful for the treatment of immune signs. In addition, the antibodies of the invention have utility as adjuvants in the treatment of genes. In particular, the antibodies of the invention may be administered before, concurrently or after administration of a vector (containing therapeutic DNA) to interfere with or reduce the host humoral response to the vector. The disease is a CD4-related disease.

As described in more detail in the Examples, CE9.1 recombinant antibodies were generated by grafting antigen binding variable Fv domains from cynomolgus monkeys into human constant regions, eg, IgG1 constant domains. More specifically, the CE9.1 antibody contains a human gamma 1 domain, a lambda constant domain. CE94, CE94K, CE94E and CE94PE contain gamma 4 constant domains or modified forms thereof, and lambda or kappa constant regions. The resulting recombinant antibody sequence is indistinguishable from human immunoglobulin sequences. As a result, these antibodies and other CD4 antibodies are prepared by similar methods and, when administered in vivo in humans, reduced immunogenic or non-immunogenic, and similar murine monoclonal compared to mouse-human chimeric antibodies derived from CD4. Local or lower serum clearance.

The CE9.1 antibody binds to human domain 1 but does not bind to CD4 in macaques, which is a region involved in interacting with MHC class molecules on antigen expressing cells. The analysis also demonstrates that other exemplified antibodies contain the same antigen binding properties as CE9.1.

Potential immunomodulatory activity was observed using CE9.1 antibodies both in vivo and ex vivo. Given these properties, i.e., those antibodies having reduced immunogenicity, lower serum clearance and potential immunoregulation compared to murine or other known anti-human CD4 mAbs derived from rodents and other antibodies described herein Is suitable for long term treatment of diseases in which immunosuppression is desired, such as autoimmune diseases and chronic inflammation such as rheumatoid arthritis. However, the antibody can be used for many other diseases, such as Hashimoto's thyroiditis, type 1 myxedema, thyroid intoxication / Grave's disease, pernicious anemia, autoimmune atrophic gastritis, autoimmune heart infection, Addison's disease, early menopause, type Diabetes mellitus, Goodpasture syndrome, myasthenia gravis, multiple sclerosis, male infertility, vulgaris ulcer, ileal swelling, sympathetic ophthalmitis, phacogenic uveitis, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, idiopathic leukopenia leucopenia), primary biliary cirrhosis, active chronic hepatitis (HBs Ag negative), latent cirrhosis, inflammatory bowel disease syndrome, Sjogren's syndrome, psoriasis, rheumatoid arthritis, dermatitis, scleroderma, mixed connective tissue disease, discus It is expected to be useful for the treatment of diseases including lupus erythematosus, systemic vasculitis and systemic lupus erythematosus (SLE).

As discussed above, rheumatoid arthritis (RA) is an inflammatory disease of the synovial fluid that includes one sign of an autoimmune phenomenon that leads to the erosion, deformation and destruction of the joint. For most autoimmune diseases, the etiology of RA is not well defined, but is characterized by increased levels of activated CD4 ⁺ T-lymphocytes in affected joints. Currently, there is no cure for RA, and treatments that debilitate the disease are designed to provide relief of symptoms and improvement in functional capacity over only a short period of time. In addition, second and third line immunosuppressants, and steroids such as azathioprine, methotrexate, and prednisolone amide in underlying diseases are provided only in more severe cases, and when used for chronic treatment, are usually slightly effective or acceptable. Toxic toxicity is not possible. In contrast, the fact that major antibodies exhibit reduced immunogenicity, longer half-lives, and potent immunomodulatory activity compared to other known anti-human CD4 mAbs derived from murine or rodents is provided for prolonged and chronic administration. It is expected to be appropriate.

In essence, the recombinant anti-CD4 monoclonal antibodies described in the above exemplified applications or other antibodies prepared according to the invention, which are incorporated by reference herein, can be used during CD4, especially during acute conditions of autoimmune diseases such as rheumatoid arthritis. ⁺ Therapeutic activity will be modulated by stopping or modifying the disruptive activity of the cells. Thus, the administration of antibodies according to the present invention is prolonged against damaging antigens (or specific tissues) that maintain the underlying disease without compromising normal host defenses against immunologically unresponsive (anergy) or opportunistic infections. Will induce resistance. Apart from RA, CD4 monoclonal antibodies are beneficial for the treatment of the same disease, and for the treatment of insulin dependent diabetes, systemic lupus erythematosus, cirrhosis, inflammatory bowel disease, multiple sclerosis and other autoimmune diseases. Specific applications are possible. In addition, they are useful for the treatment of leukemia lymphoma graft versus host disease, transplant rejection, asthma and HIV.

Recombinant anti-CD4 monoclonal antibodies prepared according to the present invention can modulate the desired in vivo therapeutic effect through one or more of the following mechanisms:

1) blocking the interaction of MHC class 2 molecules with CD4;

2) down regulation of cell surface CD4;

3) inducing anergy and / or apoptosis;

4) removal of CD4 cells; or

5) induction of resistance to autoantigens.

Although transient clearance of CD4 ⁺ cells leads to immunosuppression and possibly normalization of other overactive immune systems, the main mechanism by which anti-CD4 antibodies exert their in vivo effects is not necessarily dependent on T cell clearance. Rather, antibody binding to CD4 molecules is believed to interfere with helper T cell activity by antigen-binding antigen-specific T cell anergy or antigen binding to T cell receptors that induce resistance. For example, CE9.1 antibodies comprising human gamma 1 domains exhibit substantial immunosuppressive activity. However, chimpanzees only partially remove CD4 cells. In addition, the product in humans indicates that in clinical trials the antibody induces substantially less cell removal compared to other monoclonal antibodies.

In addition, in vivo experimental models, taga transplant specific resistance has been induced by non-removable anti-CD4 antibodies administered at the time of transplantation. Maintenance of resistance does not require anti-CD4 antibody removal but depends on the continued presence of the antigen. Simple treatment schedules with anti-CD4 antibodies will interfere with helper T cell responses to autoantigens leading to long lasting clinical improvements in the absence of generalized immunosuppression.

Based on the information contained in the main application and using known methods, those skilled in the art are stable of the exemplified recombinant anti-CD4 antibodies disclosed herein and other antibodies substantially prepared according to the invention, Resistant, effective ways can be easily identified.

As already mentioned, CE9.1 is an anti-CD4 monoclonal antibody macaque-human chimeric antibody, which is found in Chinese hamster ovary (CHO) that exhibits 91 to 92% homology with human immunoglobulin framework regions. Is an IgG1 molecule that is expressed. Thus, the molecule may exhibit a reduced immunogenic response or non-immunogenic response in humans and may exhibit longer serum half-life compared to murine monoclonal or mouse-human chimeric antibodies. In addition, antibodies exhibit limited cross reactivity with human tissue. For example, no evidence of binding of the antibody to non-lymphoid tissue was observed in the experiment. As expected, the antibody binds only to some but not all of the lymphoid cells from peripheral blood and other organs. Also, it does not react with the antibody reacts with chimpanzee CD4 ⁺ T cells, but rhesus monkeys, crab fishing monkey or pig-tailed monkeys, baboons, rats, mice, or CD4 ⁺ T cells from rabbit. Thus, based on the reactivity, chimpanzees include related species to confirm their in vivo pharmaceutical effects, ie their ability to function as an effective immunosuppressant.

CE9.1 antibodies show reversible T cell clearance activity in chimpanzees. In addition, the murine and murine / chimeric anti-CD4 monoclonal antibodies that are commonly used are likely to be improved by the reduction in potential against the expected longer half-life and immunogenic effects of these antibodies in human serum. In addition, upon administration to a human, an antibody of the invention provided with the presence of a human constant domain sequence is expected to maintain the normal effector function of the human antibody. In fact, the effector function of CE9.1 antibodies and other referenced antibodies has been evaluated in several different assays. CE9.1 antibodies have been shown to exhibit inhibited activity and weak C1q binding in mixed lymphocyte responses (MLR), but not complement mediated cytotoxicity. It also exhibits antibody dependent complement cytotoxic activity (ADDC) and has been found to bind FcR. In addition, CE9.1 antibodies were evaluated in vivo in chimpanzees where administration was found to induce partial clearance of CD4 cells and modulation of CD4 receptors.

CE9.1 antibodies were administered to chimpanzees at various doses. More specifically, the effects of 0.1, 0.3, 1, 5 and 10 mg / kg were studied in chimpanzees administered 7-14 days apart. No clinical evidence of toxicity was observed. Higher doses in mg / kg led to a reduction of 80-95% in circulating CD4 ⁺ cells 24 hours after administration. No significant inhibition of CD4 ⁺ cells was observed for 7 days after administration of 1 mg / kg. After administration of 5 mg / kg, the number of circulating CD4 ⁺ cells returned to about 40% of the base line after 7 days and to 60% of the base line after 14 days. After administration of 10 mg / kg, the circulating CD4 ⁺ cell number did not recover for 7 days after treatment, and only 40% of baseline recovered 42 days after administration. No change in other clinical pathological parameters was observed.

CE9.1 antibodies were tested in humans. For example, the activity of the CE9.1 antibody was assessed in a single dose escalation phase 1 trial in patients with rheumatoid arthritis. The results were very promising. In particular, about half of the patients administered showed an improvement of at least 30% in their weak joint scores, with an extreme positive opposite profile. In addition, as discussed above, it was initially assumed that CE9.1 was eliminated, but in fact the antibody showed only partial and transient clearance in a single dose. The partially non-removable nature of these antibodies can be advantageous because in many animal studies, it has been reported that CD4 ⁺ T cell clearance is not explicitly required for the efficacy of CD4 monoclonal antibodies. See Carteron et al., Induction of Immune Tolerance During Administration of Monoclonal Antibody to L3 T4 Does not Depend on L3 T4 ⁺ Cells, Underlying Journal of Immunology, 140: 713-716 (1988); Carteron et al, F (ab ') 2 Anti-CD4 and Intact Anti-CD4 Monoclonal Antibodies Inhibit the Accumulation of CD4 ⁺ T Cells, CD8 ⁺ T cells and BTT Cells and B cells in the Kidneys of Lupus -Prone NZB / NZW Mice , Clinical Immunology Immunopathology, 56: 373-383 (1990)]. As such, the antibody comprises a) blocking interaction of CD4 with its counterpart receptor MHC; Or) modulate CD4 on the cell surface to act like conventional receptor antagonists. Under these conditions, CD4 ⁺ T cell responses that require participation of the CD4 receptor will be reduced or blocked. It is useful that the CE9.1 antibody of the present invention shows little elimination activity in humans because it improves stability, can eliminate frequent monitoring of CD4 ⁺ cell numbers, and can also improve efficacy Because.

CE9.1 antibodies are designed to reduce CD4 cell numbers in vivo through Fc receptor and complement binding mechanisms. Studies in chimpanzees indicate that CE9.1 causes partial clearance of CD4 cells, with early results suggesting that cell removal in humans is reduced compared to other known CD4 mAbs. However, it is also desirable to prepare antibodies that lack clearance activity.

The utility of "non-removing" CD4 mAbs should be improved for the following reasons:

1) Removal of CD4 cells is not necessary for the efficacy of the CD4 mAb;

2) their safety is improved without CD4 cell removal;

3) good safety allows mAbs to be used early in the disease process;

4) potency improves without CD4 cell clearance; And

5) The absence of CD4 cell elimination eliminates or reduces the frequent monitoring of CD4 cell number, thus increasing the overall treatment benefits and costs.

This is supported by the fact that in many animal models, CD4 ⁺ T cell clearance is not required for the efficacy of CD4 mAb. Thus, non-removed CD4 mAb

1) blocking the interaction of CD4 relative receptor MHC with CD4,

2) CD4 modulation of cell surface, or

3) acts like a conventional receptor antagonist by inducing T cell anergy and / or apoptosis. Thus, CD4 ⁺ T cell responses that require the involvement of the CD4 receptor will be modified or blocked.

Typically, T cell responses induced by strong or high affinity antigens appear to be independent of CD4-co-receptor function and, therefore, are not effectively blocked by CD4 mAb. In contrast, T cells that once respond to an antigen (eg, an autoantigen) require CD4-co-receptor function and are therefore inhibited by CD4 mAb. Normally, strong autoreactive T cells (T cells with high affinity TCR for their antigen) are removed from the thymus by "clonal removal" and therefore never appear in the periphery. In contrast, T cells that induce an autoimmune response are considered weak autoreactive cells that deviate from the normal mechanism of peripheral resistance. Such cells, such as CD4, depend on the relationship of co-receptors for complete synthesis of the response. Thus, blocking co-receptors deprives these co-signaling of crucial co-signaling functions that induce partial activity or anergy. In addition, as mentioned above, it is also desirable to prepare chimeric antibodies specific for CD4 with greater stability (longer half-life in vivo).

For this purpose, various chimeric antibodies containing gamma 4 constant domains are synthesized. The domain was chosen because it could not explicitly bind to human complement or FC1 receptor. Thus, chimeric antibodies containing these constant domains are assumed to lack or substantially lack T cell clearance activity. In addition, several chimeric antibodies are prepared that contain known modifications of gamma 4 constant domains. More specifically, several chimeric antibodies contain the "E" modification described in Duncan et al., Nature, 332: 563-564 (1988) and Winter et al., WO 88/07089 (1988). And this modification is reported to reduce complementary and FC1 receptor binding. Such modifications include exchanging leucine for glutamic acid at position 236 (248 Carbet number) to reduce any residue Fc receptor binding. In addition, several chimeric antibodies are described by Angal et al., Mol. Immunol., 30: 105-8 (1993)]. This modification, which involves exchanging serine for proline at position 229 (241 Carbet number), improves stability (serum half-life) by stabilizing disulfate bonds between the heavy chains and improved tissue distribution for chimeric IgG4 deficiency modifications. Has been reported.

More precisely, the principle for the development of CE94 is to discard complement fixation and reduce FcR binding activity. The antibody differs from CE9.1 in that it contains a human gamma 4 constant domain (but not gamma 1). However, it is desirable that the results do not have regular or predictable properties. Indeed, the inventors have found that chimeric antibodies containing unmodified 9 have the same Fc receptor binding as 2 antibodies.

In contrast, the principle for manufacturing CE94K improves the productivity of the four structures. The antibody differs from CE9.1 in that it contains human kappa light chain rather than lambda. Evaluation of in vitro CD94 by Fc receptor binding assay measuring antibody binding to stimulated monocytes and mononuclear cell lines shows that CD94 still has significant Fc receptor binding activity. In addition, in the assay system, CE94 binding is indistinguishable from CE9.1 (gamma 1). Thus, the principle for the preparation of CE9E completely discarded any residue FcR binding across chimeric antibodies containing unmodified 4. CE9E contains a modified gamma 4 constant domain at one site (E modification). Finally, the principle for the preparation of CE94PE improves stability across chimeric antibodies containing 4 or mutations modified at one site (E modification). The antibody contains a gamma 4 constant domain modified at two sites (P and E modifications).

As discussed above, human 4 constant domains were selected as isotypes for abolition of effector function, ie removal of reactivity with human Fc receptors or C1q in vivo and for the absence of CD4 ⁺ cell wasting. These four candidates were selected and expressed in CHO cells.

Two of these candidate monoclonal antibodies were selected for a more extensive study, namely CE94E and CE94PE studies. As discussed above, both contain glutamic acid substitutions in the inserted CH2 region to remove residue FcR bonds associated with the four constant regions. In addition, CE94PE contains proline substitution in the hinge region, which tends to improve the stability of heavy chain disulfide bond interactions.

The antibodies have been found to be indistinguishable in their affinity for CD4, molecular weight, stability against pyrolysis, inhibition of MLR, lack of binding to FcR, and lack of activity in ADCC and CDC. Thus, both of these antibodies represent in vitro standards for high affinity CD4 mAb and complementary effector function without FcR.

The properties of CE9.1 and CE94PE are compared in Table 1. Reduction of Fc receptor binding is reduced less than the 1 1 containing chimeric antibody, preferably at least 30% to 80%, more preferably at least 50% to 80%, most preferably abolished entirely to bind to the Fc receptor. It is referred to as a chimeric antibody. However, as evidenced by the results with unmodified gamma 4 chimeric antibodies, the desired results are not predictable.

Table 1.

Comparison of effector features of CE9.1 and CE94PE

activation CE9.1 CE94PE In vitro MLRC1q binding CDCADCCFcR binding Yes No Yes Yes Yes Yes No No No Depletion of Chimpanzee CD4 Cells CD4 Receptor Regulation Partially None Depletion of CD4 Cells in Vivo (HuCD4 ⁺ Transgenic Mouse) CD4 Receptor Regulation Partially None ADCC = Cytotoxicity of Antibody-Dependent CellsCDC Complementary Cytotoxicity of FcR Fc ReceptorMLR Mixed Lymphocyte Response

As such, it can be seen from the above results that chimeric antibodies that bind to human CD4 and lack certain effector functions by selection of specific constant domain sequences are prepared according to the present invention.

For example, in the use of the exemplified chimeric anti-CD4 antibodies or other chimeric antibodies prepared according to the invention as immunosuppressants or CD4 modulators for the treatment of autoimmune diseases including rheumatoid arthritis, such antibodies may be used in certain diseases. It may be administered alone or in combination with other compounds suitable for the treatment of the disease. For example, the antibodies of the invention may be receptor fusions that counteract other proteins such as monoclonal antibody soluble receptor proteins against TNF-alpha, monoclonal antibodies against IL2 receptors, and CD40 / gp39 interactions. Protein, and CTLA 4-Ig in monoclonal antibodies that act against B7 / CD28 interaction. In addition, in the treatment of rheumatoid arthritis, the main antibody is another therapeutic agent, for example, rapamycin, Leflunomide, Tenidap, RS-61443 (Mycophenolate Mofetil). ), Surenyl (sodium hyaluronate), anti-TCR (V 17) peptide vaccine, Anerva X (anti-MHC vaccine), and in vitro Protein A immunosorbent or theirs It can be administered with a combination. In addition, the antibodies of the present invention may be administered with other antibodies prepared according to the present invention or specific for human CD4 and known in the art. This leads to synergistic effects, eg different binding of these antibodies with different epitopes of the CD4 protein.

The following examples are intended to illustrate the invention in more detail.

Example 1

Cloning and Expression of Monkey / Human Chimeric Antibodies Specific to CD4

The following are specific examples of the methods and antibodies of the invention.

Generation of monkey immortalized B-cell lines

Adult cynomolgus monkeys (White Sands New Mexico Primate Center) from 150 to 300 soluble CD4 (sCD4) or cell membranes (1 x 10 ⁸ cells) from CD4 positive cell line SupT1 using standard adjuvant Immunizations were repeated six times every two to three weeks, each time inoculated with 100 sCD4 in the inguinal part of one thigh of the monkey, followed by a second immunization, one week later from the same thigh. Outflow lymph nodes were surgically removed Tissues were cut and rinsed with sterile DMEM medium to remove lymphocytes from lymph nodes Cell suspensions were filtered with nylon gauze and collected by centrifugation at 100 xg for 10 minutes.

About 1 × 10 ⁸ lymphocytes were suspended in tris-ammonium chloride buffer (16 mM, pH 7.5) and warmed at 37 to 5 minutes to lyse erythrocytes. Lymphocytes were collected by centrifugation, resuspended in L-leucine methyl ester (LME) and incubated for 37 to 45 minutes. LME treated cells were filtered through a nylon screen and centrifuged. 1 ml of calf fetal serum was added, cells were suspended and washed twice with serum-free RPMI. The cell number was counted and mixed with the same number of K6H6 / B5 heteromyeloma cells washed twice in medium without serum in 50 ml conical centrifuge tubes. The cells were appropriately suspended by slow addition of 1 ml of 50% PEG (polyethylene glycol) over 1 minute with gentle stirring. Cells were then resuspended by adding 20 ml of serum free medium over 5 minutes with dilution with PEG and light mixing. After washing twice with serum free medium, cells are resuspended in RPMI medium containing 20% calf fetal serum and gentamycin at a concentration of 5 x 10 ⁵ /0.1 ml and placed into 96 well micro tissue culture plates. Placed at 0.1 ml per well. Eastern blood HAT medium (0.1 ml) was added to each well and allowed to hybridize by growing for 14-17 days prior to screening.

Screening of Fused Cell Hybrids for the Production of Anti-CD4

The assay to determine anti-CD4 specificity is as follows: ELISA plates were coated with recombinant sCD4 at a concentration of 100 ng per well and blocked with 1% bovine serum albumin in PBS. Fifty aliquots of hybridoma supernatants were removed from each well and incubated in sCD4 coated plates for 60 minutes. Binding was detected by incubating ¹²⁵ I labeled goat anti-human or goat anti-monkey Ig for 60 minutes. After washing four times with distilled water, the wells were counted with a gamma counter. Positive wells with replicated and hybridoma cells were reanalyzed from the wells subcloned three times, first with 5 cells per well and then twice with 1 cell per well. At this stage, anti-sCD4 positives were screened for their ability to bind cell surface CD4. This was done by inhibiting binding of the anti-CD4 murine monoclonal, term 1F3, to the CD4 positive cell line supT1. Simply by co-incubating 10 ¹²⁵ I-labeled 1F3 with 10 different amounts of monkey anti-CD4 and 3 × 10 ⁵ supT1 cells / well. After incubation for 1 hour at room temperature (about 20-25), it was removed by vacuum on a glass fiber filter. After extensive washing with PBS, the filters were counted with a gamma counter to measure 1F3 binding inhibition to supT1 cells by monkey hybridoma supernatant.

Candidate clones were prepared that produced antibodies exhibiting strong inhibition against 1F3. Clones were isotyped using human isotyping agents and were found as IgG2 with lambda light chains. The cell lines were grown to larger numbers for the cloning of their immunoglobulin genes.

Cloning of heavy and light chain variable region genes from monkey immortalized B-cells

Total RNA was isolated from 1 × 10 ⁷ monkey immortalized B-cells using guanidinium isothiocyanate method. One tenth of the total RNA was used to make single stranded cDNA using oligo-dT oligonucleotide primers and reverse transcriptase. One tenth of the total amount of single stranded cDNA was used to perform the PCR reaction. The six PCR reactions comprise one of six 5 'V _H -based specific oligonucleotide primers containing Sal restriction sites with IgG 3' constant region oligonucleotides each containing a Nhe site, both of which are shown in FIG. 7. It is shown at -1. Similarly, five PCR reactions were performed using one of five 5 'lambda leader sequence oligonucleotide primers containing the Bal II site and a 3' lambda constant region primer containing the Avr II site. The reaction conditions are as described above. Each PCR reaction was performed in triplicate. Each product of the heavy and light chain amplification reactions was performed on a 1.2% agarose gel. VH4 heavy chain primer (5'-ACTAAGTCGACATGAAACACCTGTGGTTCTT-3 ') and lambda primer (5'-ATCACAGATCTCTCACCATGACCTGCTCCCCTCTCCTCC-3') provide a strong band in agarose gel electrophoresis. The product of the reaction was used for cloning into vector TCAE 6 containing human IgG1 and human lambda constant region sequences.

Cloning of two variable region genes into expression vector TCAE 6 was performed one after the other. First, the heavy chain PCR product and vector TCAE 6 were enzymatically digested with restriction enzymes Sal and Nhe , the product was extracted with phenol / chloroform and passed through a SEPHADEX G-25 spin column. PCR products were ligated with the cleavage vector overnight at 14 in the presence of T4 DNA ligase. About 500ng of total DNA was ligated to a volume of 10 with an insert / vector molar ratio of 10: 1. The ligated material was used to transform XL-1 blue competent cells (Stratagene), and the transformed cells were placed on LB agar plates containing 50 / ml ampicillin. Colonies of ampicillin resistant bacteria were screened out and cultured as 5 minicultures. Plasmid DNA was extracted from each culture by standard alkaline lysis, digested with restriction enzymes Sal I and Nhe I, and the product was placed on a 1.2% agarose gel. Plasmids with an insert of about 450 bp were used as template for subsequent cloning of the light chain variable region. The light chain CPR reaction product and the product containing the light chain insert were cleaved with restriction enzymes Bgl II and Avr II and ligated together. Plasmid minicultures were screened by cleavage with Bgl II and Avr II. Enzymes that gave an insert of about 400-450 bp were marked positive. Plasmids containing both Sal I / Nhe I and Bgl II / Avr II inserts were incubated in higher amounts for DNA alignment.

Serial chimeric antibody expression vectors TCAE 5.2 and TCAE 6 were derived from the vector CLDN, which is a derivative of the vector RLDN10b (253 Science , 77-79 (1991)) itself. RLDN10b is also a derivative of the expression vector TND (7 DNA , 651-661 (1988)).

RLDN10b is different from vector TND in the following manner. Place the dihydrofolate reductase (DHFR) transcription cassette (promoter, cDNA and polyadenylation region) between the tissue plasminogen activator cassette (t-PA expression cassette) and the neomycin phosphotransferase (NEO) cassette. All three cassettes were in series and in the same transfer orientation. In the CLDN, the DHFR gene promoter was replaced with the mouse beta globin major promoter (3 Mol. Cell Biol ., 1246-54 (1983)) and t-PA cDNA was replaced with polylinker. All three eukaryotic transcription cassettes (expression, DHFR, NEO) can be isolated from bacterial plasmid DNA (pUC9 derivatives) by digestion with restriction endonuclease Not I.

CLDN differs from RLDAN10b because the Rous LRT in front of the polylinker has been replaced by the human cytomegalovirus adjacent early gene promoter enhancer (41 Cell , 521, (1985)).

Expression vectors TCAE 5.2 and TCAE 6 differ from CLDN in the following ways:

1) The vector is

a) Human immunoglobulin light chain constant region induced by amplification of cDNA by polymerase chain reaction. In TCAE 5.2, the region is a human immunoglobulin light chain kappa constant region (Kabat amino acid number 108 to 214, allotype Km3), and in TCAE 6 the human immunoglobulin light chain lambda constant region (Kabat amino acid number 108 to 215, genotype Oz minus, Mcg minus, Ke minus allotype).

(b) human immunoglobulin heavy chain constant regions; In both constructs the human immunoglobulin heavy chain is the gamma 1 constant region (Kabat amino acid number 114 to 478, allotype Gm1a, Gm1z), which region is induced by cDNA amplification by polymerase chain reaction.

(c) DHFR; Containing its own eukaryotic promoter and polyadenylation region.

(d) NEO; It contains four transcription cassettes (instead of three), such as containing their own eukaryotic promoter and polyadenylation region, in serial order.

2) Human immunoglobulin light and heavy chain cassettes contain synthetic signal sequences for the secretion of immunoglobulin chains.

3) Human immunoglobulin light and heavy chain cassettes contain specific DNA linkers that maintain the transcriptional reading frame and allow insertion of immunoglobulin variable regions of the light and heavy chains that do not alter the amino acids commonly found in immunoglobulin chains. The incorporation of the above-described variants allows the construction of the vectors TCAE 5.2 and TCAE 6. Cloning of the immunoglobulin light and heavy chain variable region genes from the anti-CD4 heterohybridoma cell line E 9.1 to TCAE 6 results in a construct deposited with the ATCC. The construct, deposited for the CE9.1 antibody, contains a cynomolgus monkey immunoglobulin heavy chain variable region and a cynomolgus monkey immunoglobulin light chain variable region, the sequences of which are described in FIGS. 1 and 2, respectively, Cloned from CD4 hybridoma cell line E9.1. The heavy chain constant region is of human origin of gamma 1 isotype and Gmla, Gmlz allotype. Lambda light chain constant regions are also of human origin of Oz minus, mcg minus genotype and Ke minus allotype. The immunoglobulin gene is cloned into the mammalian expression vector TCAE 6 described in the above application, which is incorporated herein by reference, and a monkey / human anti CD4 chimeric antibody is produced when electroporated with mammalian cell line CHO. The DNA constructs described herein have been used to transform bacterial strain XL-1 blue, selected from antibiotic ampicillin and deposited with bacterial cell suspension in sterile LB medium containing 15% glycerol.

DNA sequencing

Plasmid DNA was prepared from 100 cultures. A further mixture of 2.5M sodium chloride and 20% polyethylene glycol (6 vol) was further purified by precipitating on ice for 15 min (1 vol). After centrifugation at 10,000 × g for 20 minutes, the pallet was washed with 70% ethanol, recentrifuged and dried in Speedivac (Savant). Pallets of DNA were resuspended in deionized water at a concentration of 150-250 /. Sequencing was performed with 5 double stranded DNA using Sanger's technique. An array primer homologous to the upstream and downstream sequences of the expression vector of the light or heavy chain insert was used. The insert was sequenced bidirectionally from 5 'to 3' and from 3 'to 5'. Two clones of the anti-CD4 light chain and two clones of the anti-CD4 heavy chain, respectively generated from each PCR reaction, were arranged in parallel to determine whether nucleotide variants were introduced during the PCR reaction. The two heavy and two light chain clones selected were found to be identical over the entire length, which would confirm that there were no errors during the amplification process. The sequences of the anti CD4 heavy and light chains are shown in FIGS. 1 and 2.

Expression of Monkey / Human Chimeric Anti-CD4

The expression vectors TCAE 5.2 and TCAE 6 are not only used for stable expression of all into the cell lines Sp2 / 0 and CHO, but can also be transiently expressed in the cell line COS since they contain SV40 origin. COS cell expression was performed as follows: seeding COS cells one day before transfection, to form 50-70% confluency the next day. The medium was removed and the cells washed twice with transfection buffer (TB-140 mM NaCl, 25 mM Tris, 5 mM KCl, 0.5 mM Na ₂ HPO ₄ , 1 mM MgCl ₂ , 1 mM CaCl ₂ ). 30 cesium chloride purified TCAE 6 plasmids containing anti-CD4 monkey / human chimeric heavy and light chain immunoglobulins were mixed with 3 DEAE dextran (1 / of TB) per dish. DNA was incubated with the cells at 37 for 1 hour. The DNA solution was removed and replaced with 3's 20% glycerol for 1.5-2.5 minutes and the cells washed twice with TB. Cells were incubated in 5 fresh media containing 37 to 100 uM chloroquine for 3 to 5 hours and then washed twice with media and incubated with normal DMEM for 72 hours. Supernatants 100 from transfected COS cells were assayed at various dilutions for the presence of antibodies by a technique based on ELISA. Goat anti-human lambda was used to coat 96 well assay plates under standard ELISA conditions and peroxidase-labeled goat anti-human IgG was used as detection antibody. COS cells have been found to produce 10-40 ng / monkey / human chimeric antibody. A higher volume of supernatant was concentrated 10-fold and used for direct binding of RIV to CD4 positive SupT1 cells. The original whole monkey antibody and unrelated human immunoglobulins were used as positive and negative controls, respectively. In addition, monkey anti-CD4 and monkey / human chimeric anti-CD4 were used to inhibit binding of high affinity mouse anti-CD4 (1F3) antibodies. The results indicate that the monkey / human recombinant antibody (ATCC No. 69030) not only binds to CD4 positive cells but also inhibits binding of 1F3 to CD4 positive cells at nearly the same concentration of whole monkey antibody or 1F3 itself. It was.

Example 2

This example includes the effect of CE9.1 on T cell proliferation and IL-2 production in MLR, Fc receptor and complement binding properties of CE9.1, and the ability of CE9.1 to mediate ADCC and CDC responses. To the ex vivo properties of CE9.1. In addition, in vivo effects on CD4 receptor mediated and lymphatic subsets in peripheral blood were analyzed. The following materials and methods were used in this Example [Reference: Anderson et al, "In vitro and in vivo characterization of a primatized mAb to human CD4: mAb caused CD4 receptor modulation but not CD4 T cell depletion in chimpanzees "].

Materials and methods

Molecular Structure and Expression of PRIMATIZED® Anti-CD4

The variable region immunoglobulin gene was amplified by PCR and cloned from heterohybridoma derived from monkeys immunized with sCD4 as described above. [Newman, RA, et al, "Primatization of recombinant antibodies for immunotherapy of human disease: a macaque / human chimeric antibody against human CD4 ", Biotechnology, 10: 1455 (1992). Heavy and light chain variable region genes were inserted in a cassette expression factor, ie TCAE 6, in a serial fashion and expressed as IgG1 after stable integration into DHFR ^- CHO cells. See Newman, supra. Three amplifications in increasing amounts of methotrexate allowed the cell line to develop to express 750 ug / excess over 8 days. Generation of cell lines that grow in suspension culture and gradually expand before injecting into the hollow fiber reactor occurred [Ref .: Evans et al, “Large-scale production of murine monoclonal antibodies using hollow fiber bioreactors”, BioTechniques 6 (8). ): 762 (1988).

mAb CE9.1 was purified by passing the culture supernatant from the medium at a rate of 125 / min through a Prosep A column (300, Bioprocessing Inc.) previously equilibrated with phosphate buffered saline (pH 7.2). The column was washed with PBS until a baseline was established and the bound antibody eluted with 5 column volumes of 0.2 M acetic acid / 0.1 M glycine buffer (pH 4.0). The recovery was about 90%. The eluate was brought to pH 5.5 and passed through a Q-Sepharose column (Pharmacia). CE9.1 was bound to the column washed with 25MM Tris-HCl, pH 8.5. The antibody was eluted with 50 mM Tris-HCl, pH 6.5 containing 100 mM NaCl, and concentrated by defiltration (Millipore Pelican) against normal saline for USP injection. CE9.1 was finally filtered through a 0.04um Nylon ₆₆ NDP filter (product of Pall Filtration).

Binding specificity: CD4 ⁺  Binding of CE9.1 to SupT-18 Cells

96-well U-bottom microliter plates (cloning) were preblocked with PBS containing 0.2% bovine albumin and 0.1% sodium azide for 1 hour on ice. SupT-18 cells (1 × 10 ⁵ ) pre-washed with the same buffer were incubated with CE9.1 (2.4 pg / ml-10 / ml) with varying concentrations over 30 minutes on ice. The cells were washed twice and incubated with a second antibody (FITC-labeled goat anti-mouse Ig) for 30 minutes on ice. Cells were washed twice, resuspended in fixed buffer (2% formaldehyde in PBS) and analyzed using a FACScan flow cytometer (Becton Dickinson).

Binding specificity: analysis by flow of binding of CE9.1 to human peripheral blood leukocytes

Mononuclear cells were isolated from human peripheral blood using standard "Ficoll / Hypaque" centrifugation techniques [Boyum A. "Separation of blood leukocytes, granulocytes and lymphocytes", Tissue Antigens 4: 269 (1974). The interfacial layer containing peripheral blood mononuclear cells (PMNC) was removed, washed with Hank's Balanced Salt Solution (HBSS) and counted. 5 × 10 ⁶ cells were incubated at 20 CE9.1 (25 /) for 4 to 30 minutes. Cells were washed with HBSS and incubated with 20 goat anti-human IGG-EITC (Fisher Scientific). After an additional 30 minutes of incubation on ice, cells were analyzed on a Becton Dickinson FACScan instrument using pre-measurement with auto compensation and calibrite beads. Viable lymphocyte populations were identified by an anterior-to-right light scatterer and whole lymphocyte populations were isolated by gating all other events. Subsequent fluorescence measurements only reflect gated lymphocyte events. MAbs used for quantification of double contaminated cells and for subsequent studies on chimpanzee blood include anti-human CD3 (Leu-4-FITC; Becton Dickinson); Fluorescein-conjugated anti-human CDS (Leu-2a-FITC; Becton Dickinson); Phycoerythrin-conjugated anti-human CD8 (Leu-2a-PE; Becton Dickinson); Phycoerythrin-conjugated anti-human CD20 (Leu-16-PE; Becton Dickinson); Fluorescein-conjugated goat anti-human IgG F (ab ') 2 (capel); And phycoerythrin-conjugated murine animal anti-CD4 (OKT; Ortho Pharmaceuticals).

Human tissue cross reactivity

CE9.1 was evaluated for cross reactivity to normal human tissue. Biotinylated CE9.1 was tested for cryostat cleavage frozen fragments from 32 different tissues using the avidin-biotin immunoperoxidase technique [Wilchek, M. et al, "The avidine-biotin complex in bioanalytical application ", Anal. Biochem., 171: 1 (1983). SupT1 cells (CD4 ⁺ ) were used as positive controls and SB cells (CD4-) were used as negative control cell lines. Irrelevant biotinylated mouse / human (IgG) chimeric antibodies were used as negative antibody controls.

For most tissues, three separate specimens were tested and the reactivity with CE9.1 was recorded for 0-3 ⁺ . In some tissues, different structures within the tissue were recorded separately. For example, in the liver, hepatocytes, bile ducts and Kupffer cells were recorded independently.

Species specificity

Peripheral blood from several common laboratory primates and non-primates were screened with CE9.1 to confirm possible cross reactivity of CD4 positive T cells. This group includes chimpanzees, baboons, rhesus monkeys, cynomolgus monkeys, and pigtail macaque rats, mice, rabbits, and dogs. Blood cells were isolated from whole blood 1-5 by centrifugation (1500 rpm for 5 minutes) at 4 and washed by resuspension in equal volume of PBS. This procedure was repeated one more time and the cells were resuspended in the same volume of fetal bovine serum. Cell suspensions 200 from each species were placed in 15 conical centrifuge tubes with a CE9.1 of 25/25. Antibodies and cells were mixed and placed on ice for 20 minutes and then washed evenly with HBSS. Then 20 ul goat anti-human IgG-FITC (Fisher Scientific) was added and the samples mixed. After an additional 30 minutes of incubation on ice, the samples were separated from the ice and 10 cell lysate buffers (0.01 M potassium bicarbonate, 0.17 M ammonium chloride and 0.1 M sodium EDTA, pH 7.4) warmed to 37 Added. Samples were incubated at room temperature for 15 minutes and centrifuged at 1500 rpm for 5 minutes. Labeled cell pellets were further washed twice in HBSS (pH 7.4) containing 1% bovine serum albumin and 0.05% sodium azide. Labeled cells were fixed by resuspending in fixed buffer (0.5 M sodium chloride with 1.0% formaldehyde; filtered through a 0.22 um filter). Samples were analyzed on a Beckton Dickinson FACScan instrument as described above.

In Vitro Functional Analysis: Lymphocyte Response Mixed in One and Three Ways

Human or chimpanzee T cells (1.3 × 10 ⁵ ) were mitomycin C treated PBMCs (6.0 × 10 ⁴⁾ obtained from unrelated donors of human or chimpanzee origin, respectively, with or without CE9.1 in flat bottom microwells. ) Was incubated for 7 days. Tritiated thymidine 1 uCi / well was added to the culture during the last 18 hours of incubation. Microtiter plates were centrifuged and the cell pellet was washed with HBSS and counted with a liquid scintillation counter. Each sample was assayed three times.

Human MLR was performed using three separate and unrelated donors as simulators and responders. This protocol was modified to maximize the likelihood of good response in HLA-uncharacterized random samples of red cross pale yellow coat blood. In this protocol, none of the donor blood was treated or irradiated with mitomycin C.

THP-1 cell adhesion assay to assay Fc receptor binding activity of CE9.1

This assay relies on bridging two cell lines, with anti-CD4 antibodies, one cell expressed CD4 and the other cell expressed Fc receptor. The CD4 expression partner used is the adherent murine fibroblast line DAP (DAP / CD4) transformed with human CD4. Fc receptor containing cells are THP-1. DAP / CD4 cells were placed in 96-well flat bottom plates (100 ul / well; 25,000 cells / well) and fixed overnight. THP-1 cells were resuspended in RPMI medium (1 × 10 ⁶ cells /) 50 and induced for 37 to 24 hours by addition of IFN 50U /.

IFN-induced THP-1 cells were loaded with calcined acetomethoxy ester (CAM; Molecular Probes) as follows: Cells were loaded with Dulbecco with loading buffer (calcium and magnesium and 0.1% bovine serum albumin). Dulbecco) PBS) and resuspended at 5 × 10 ⁶ cells / in the same buffer 10. CAM (1 / in DMSO) was diluted with loading buffer (1:50) and added to THP-1 cell suspension (1: 1 v / v). After 20 minutes of incubation at room temperature, fresh loading buffer 25 was added to each cell / CAM mixture 4 and incubated for another 40 minutes at room temperature. Cells were washed twice with loading buffer and resuspended at 8 × 10 ⁶ cells /. Serum dilutions of CE9.1 in PBS (without calcium, magnesium or BSA) were added to the wells containing CD4 ⁺ DAP cells and incubated for 5 minutes at room temperature. 50ul of CAM loaded THP-1 cell suspension was added and the plates were incubated for 1 hour at room temperature in the dark. Control wells containing no DAP cells were also assayed. After incubation, the wells were washed three times with PBS. After the final wash, 100ul of PBS per well was added followed by 10ul of 20% Triton X-100. After placing on a shaker for 10-15 seconds, the plates were read on Fluoroscan (MTX Lab systems Inc.).

Activated Mononuclear Cell Binding Assay to Measure Fc Receptor Binding Activity of CE9.1

Fc receptor assays were performed as described for THP-1 cells, except for the following differences. Monocytes were prepared from fresh human peripheral blood by standard Ficoll / Hypaque and Percoll concentration gradient separation. Monocytes were stimulated with IFN as above but for 48 hours. Plates were coated with stimulated mononuclear cells for 24 hours, in which the CD4 ⁺ lineage SupT-18 was loaded with CAM as described above. SupT-18 cells were added to plates coated with mononuclear cells stimulated as described above. The main difference in this assay is that CD4 ⁺ cell line CAM is loaded and added to FcR containing cells on the plate. In the above assay using THP-1 cells, the order was reversed.

Binding to FcRII Transfected Murine Fibroblasts

Murine fibroblast line (CDW32-L) transfected with human FCRH was obtained from ATCC. Direct binding of CE9.1 was measured by incubating the antibody in the presence and absence of sCD4. Binding of CE9.1 was detected by incubation with goat anti-human Ig-antibodies conjugated to horseradish peroxidase (Southern Biotech). Fab fragments of CE9.1 were generated by enzymatic digestion and used as negative controls. Absorbance obtained from CE9.1 (and Fab fragments) preincubated with cells in the presence of sCD4 was subtracted from the absorbance obtained for the antibody in the absence of sCD4.

ADCC assay (lysate of supT1 cells)

Fresh heparinized human blood samples were collected and the PMNC was isolated by standard centrifugation process on Ficoll / Hypaque. Erythrocytes in a pale brown coout were lysed with ammonium chloride buffer and cells were washed twice in Hank Balance salt solution. Peripheral blood lymphocytes (PBL) were stimulated with 10 units of IL-2 per RPMI / 10% calf fetal serum (FCS) for 24 hours at 37, 5% CO ₂ . After 24 hours, PBLs were resuspended in RPMI / 5% FCS.

SupT1-18 cells (1 × 10 ⁶ ) were labeled by incubating with 100 uCi ⁵¹ Cr for 1 hour at 37, 5% CO ₂ . Cells were washed twice with RPMI / 5% FCS and 1 × 10 ⁴ cells were added to each well. Three CE9.1 antibodies were serially diluted 1: 2 with RPMI / 5% FCS and aliquots were added three times to SUPT1-18 containing wells for 30 minutes at 37, 5% CO ₂ . 100 uL of 1% Triton X-100 and 100 uL of medium were used as the maximum and spontaneous release controls, respectively. IL-2 stimulated PBL (8 × 10 ⁵ cells) was added to the wells. Plates were centrifuged at 900 rpm for 30 minutes and incubated for 16 hours at 37, 5% CO ₂ . Supernatants from each well were collected and the amount of radioactivity counted in a gamma counter. The assay was performed three times. Cell lysis was measured using the following equation:

Dissolution rate% = [(sample factor-spontaneous release) / (maximum release-spontaneous release)] X 100

Clq linkage assay

Clq assay was performed using SupT1-18 CD4 positive cells in a suspension of 4 × 10 ⁶ /. Equivalent concentrations of 20ug / of CE9.1 and control affinity purified monkey anti-CD4 antibodies (50ul) were added to 2 × 10 ⁵ CD4 positive target cells. Cell suspensions and antibodies were incubated for 1 hour on ice and then washed twice with 1% BSA in PBS. 50 uL of human Clq (10 ug /) was added to each tube and incubated for 1 hour on ice. Each tube was washed twice and then incubated with a 1:15 dilution of rabbit anti-human Clq FITC (50 uL) (in the dark, 1 hour on ice). The cells were washed again twice and fixed in 0.5 1% formaldehyde / PBS. Cells were analyzed in a Beckton Dickinson FACScan flow cytometer using Consort 30 software for data acquisition and analysis.

Complement Mediated Cytotoxicity Assay

SupT1-18 cells (1 × 10 ⁶ ) were labeled by incubating with 100 uCi ⁵¹ Cr for 1 hour at 37, 5% CO ₂ . Cells were washed twice with RPMI / 5% FCS and 1 × 10 ⁴ cells were added to each well. CE9.1 and control anti-CD4 antibodies were serially diluted 1: 2 with RPMI / 5% FCS and aliquots of 50 ul were added three times to the SUPT1-18 containing wells. 100 uL of 1% Triton X-100 or 100 uL of medium was added to the wells to determine the maximum and spontaneous release of ⁵¹ Cr, respectively. After 90 minutes of incubation at 37, 5% CO ₂ , a 1: 5 dilution of rabbit complement (Cappel) was added to the wells. The plate was incubated for another 90 minutes at 37, 5% CO ₂ , then centrifuged at 900 rpm for 30 minutes. Supernatants from each well were collected and the amount of radioactivity counted in a gamma counter. The assay was performed three times. Cell lysis was measured using the following equation:

Dissolution Rate% = [(Sample Factor-Spontaneous Release) / (Maximum Release-Spontaneous Release)] X 100

In vivo Studies of Chimpanzees

Six chimpanzees were divided into three groups of two each: group I (salt control), group II (CE9.1 antibody 10.0 /) and group III (CE9.1 antibody 10.0 /). Group II animals were retreated to CE9.1 10.0 / after 30 days, except that their count of CD4 ⁺ T cells was restored to 30% of baseline. Group III animals were retreated to CE9.1 10.0 / after 30 days, with the counts of their CD4 ⁺ T cells restored to 70% of baseline. If these values have not been reached by 30 days, animals are screened for CD3 ⁺ , CD4 ⁺ and CD8 ⁺ T cell values at 2 week intervals until CD4 ⁺ T cell values reach their respective target values for the group. It was. At this time, animals were administered intravenously with CE9.1 antibody 10.0 / until the three doses were maximized again.

Baseline measurements of total leukocyte counts, lymphocyte and granulocyte values, and CD3 ⁺ , CD4 ⁺ and CD8 ⁺ lymphocyte subgroups were performed on days −6 and 0 immediately prior to administration, and again 24 hours and 14 days after administration. Three treatment cycles by 10 / dose were each administered to the chimpanzees in this study.

result

Binding specificity of mAb CE9.1

Affinity measurements by SPR for binding of CE9.1 to soluble CD4 showed that the Kd was 1.0 nM [Brigham-Burke et al. North American BIAsymposium 1995 (in press)]. Binding to CD4 ⁻ cell lines did not appear, and inhibition studies demonstrated that binding to CD4 ⁺ cells could be completely inhibited by soluble CD4 ⁺ in a stoichiometric manner.

To determine the specificity of CE9.1 reactivity, binding to freshly isolated human PBMCs was measured by dual color flow cytometry analysis. 9 shows that about two thirds of CD3 ⁺ cells bind to CE9.1. Within the lymphoid subgroup, all cells that bind to OKT4 are also positive for CE9.1, while CD8 ⁺ cells are all negative. Although the state of reactivity is not clearly determined, some CD3- cells also exhibit reactivity with CE9.1.

To measure the tissue reactivity of CE9.1 including 32 different normal human tissues of lymphoid and nonlymphoid origin, immunohistochemical analysis was performed. Nonlymphoid tissues include most organs, brain, heart, skeletal muscle skin, liver, kidney, gland and regenerative tissue. This analysis does not show cross-reactivity to any tissue that differs from tissues of lymphatic origin, including lymph nodes, spleen, tonsils and peripheral blood (data not shown). Stains defined by lymphoid aggregates were also observed in many intestines, lungs, esophagus and skin.

Inhibition of human MLR by CE9.1

The effect of CE9.1 on the T cell response was assessed by human MLR as an IL-2 production or proliferative response. CE9.1 inhibited both proliferation and IL-2 production with an IC ₅₀ of 10-30 ng /, showing about 80% inhibition at 60 ng / (FIG. 10).

Fc Receptor Binding Activity of CE9.1

Cell-cell adhesion assays based on CD4 and Fc receptors have been developed to measure the reactivity of CE9.1 with Fc receptors on monocytes. In one assay configuration, monocytes were isolated from fresh PBMCs by percoll gradient centrifugation, seeded in microliter plates and stimulated with IFN for 48 hours. After 48 hours, dye loaded CD4 ⁺ SupT1 cells were added to activated adherent monocytes with or without CE9.1. In a second configuration of this adhesion assay, mononuclear non-adherent cell lines, ie THP-1 cells, were stimulated with IFN. After 24 hours, activated THP-1 cells were loaded with marker markers and adhered to adherent CD4 ⁺ fibroblast transformants previously plated (24 hours ago) in microliter plates with or without CE9.1. Added. In both cases, cell-cell adhesion is dependent on binding of mAb to CD4 on one cell and Fc receptor on the other.

The data provided in FIGS. 10A, 10B and 10C, based on IFN activated new monocytes and CD4 ⁺ SupT1 T cells, is about 20 ng / ED ED ₅₀ , CE9.1 mediates cell-cell adhesion in a dose dependent manner. Shows. Attachment is completely inhibited by sCD4 and cannot be mediated by the F (ab ′) 2 fragment of CE9.1. Monocytes that are not activated by IFN are unable to bind CE9.1 (FIG. 10B). Similar data are also obtained with the assay based on THP-1 and CD4 ⁺ fibroblast assays (data not shown). Murine fibroblast cell lines transformed with human FCRII receptors are also observed (data not shown).

Antibody-Dependent Cell Mediated Cytotoxicity (ADCC)

Radiolabeled SupT1 cells used as targets in ADCC assays have been demonstrated to be specifically lysed by effector cells in the presence of CE9.1. Maximal cytotoxicity is reached at about 6 ug / with about 50% total specific lysis (FIG. 12). As a positive control, murine anti-CD4 of IgG2a isotype (4D9) was used. The antibody is very similar in nature to CE9.1, providing the same level of total cell lysis. Thus, CE9.1 is very effective in binding Fc receptors to effector cells and mediating the death of CD4 ⁺ target cell lines.

Complement fixation by CE9.1

Binding of Clq was measured by flow cytometry, as described above (materials and methods). As shown in FIG. 13, despite the fact that CE9.1 contains a human heavy chain constant region of the gamma 1 subtype, this shows only minimal binding of Clq (FIG. 13). Affinity purified monkey anti-CD4 serum antibodies are effective at mediating Clq binding, suggesting that the lack of Clq binding by CE9.1 is property specific. The lack of Clq binding by CE9.1 is reflected in the impossibility to fix the complement. Affinity purified anti-CD4 antibodies and murine monoclonal IgG2a from monkey serum both result in significant lysis over the same concentration range.

CE9.1 Species Cross Reactivity

Flow cytometric analysis of lymphocytes from different species shows that only chimpanzee and human cells bind CE9.1 strongly. Bibi is the only species that exhibits weak reactivity with CE9.1 (10 times lower than humans). Human and chimpanzee lymphocytes respond equally with mAb (Table 2). This is reflected in similar inhibition of T cell proliferation and IL-2 production in chimpanzee MLR by CE9.1 antibody.

TABLE 2

 Bell  Responsive  Human Chimpanzee Baboon Rhesus Macaque Monkey Pigtail Macaque Dog Rabbit Rat Mouse   +++ +++ +-------

In vivo study in six chimpanzees

Based on the lack of elimination of CD4 cells in the expanded dose study in one chimpanzee, four chimpanzees were given a dose of 10 / (otherwise two animals in the control group were given saline). As described in Materials and Methods, two dose groups were each given 10 / on day 0 of the study. Figure 15 summarizes the effect on CD4 and CD8 counts in these animals. It was found that the cells expressing the CD4 receptor decreased immediately after the antibody administration. The decrease in CD4 counts is manifested immediately after each administration of dodge antibody. Similar variations in CD4 counts were not seen in the saline control group. CD8 counts remain unaffected throughout the course of treatment, even if variability is observed on a daily basis (FIG. 15, left panel, open source). By testing the CD3 ⁺ -CD8 ⁺ individuals, a slightly softened decrease in the number of CD4 was observed. The data suggest the appearance of CD3 ⁺ CD8 − T cell populations, which may be the result of CD4 antigen mediation. The precise regulatory mechanism is obscure at this stage, but may include internalization or divergence of Fc receptors expressed in other lymphocytes or monocytes. Comparison of cell numbers in FIG. 15 indicates that some clearance of CD4 ⁺ cells may occur, even if the main effect is due to CD4 receptor mediation. In most cases, the modulatory effect appears to last for about 7-10 days immediately after antibody administration, after which CD4 expression reverts directly below baseline.

The overall CD4 count remains lower than the baseline time point by 10 to 50%, but after stopping of treatment, it returns to the normal range. Chimpanzees are maintained for a period of up to 150 days (groups 1 and 2) or 300 days (group 3). The CD4 counts of animals in group 2 return to normal levels after 80 days of the last treatment, while animals in group 3 return to within 20% of baseline in the same time range.

Example 3

This example describes the genetic construct of a DNA expression vector used in mammalian cells to produce CE94PE, a rhesus / human chimeric anti-CD4 antibody containing human 4 isotypes comprising P and E modifications. will be.

Composition of DNA Expression Vectors

Human gamma 4 heavy chain gene was obtained from cell line TPIT10 (obtained from S. Morrison of UCLA) using 5 'IDEC primer # 479 and 3' IDEC primer # 462 (see Figure 16) containing Nhe I and BamH I sites, respectively. Isolation by PCR. Whole cloned fragments of human gamma 4 were sequenced and described by Kabat et al., NIH Publication Fifth Edition No. 91-3242, U. S. Dept. of Health and Human Services (1991)]. Nhe I / BamH I fragments were cloned into expression factor Anex 2. The entire light and heavy chain immunoglobulin genes from the plasmids were transferred to another expression plasmid on the Bgl II to Sac I fragments. The plasmid was named anti-CD4 (G4, L, Oz) in NEOSPLA3F.

PCR mutagenesis was used to alter amino acids # 229 and 236 in the gamma 4 constant region. PCR was performed using 5 'primer GE212 (Midland) and 3' IDEC primer # 698 containing Nhe II and BspH I restriction sites, respectively, and the fragments were subjected to anti-CD4 (G4, L, Oz) in three portions of ligation. Cloned into) and the sequence plasmid was named anti-CD4 (G4 (PE), L, Oz-) in NEOSPLA3F.

Example 4

Expression in Chinese Hamster Ovary (CHO) Cells

Integration of Plasmids and Screening of Antibody Producing Clones

CHO cells (DG44) were grown in CHO-S-SFM (GIBCO / BRL, CHO cells) containing 50 uM hypoxanthine and 8 uM thymidine (GIBCO / BRL, CHO medium). See also Urlaub et al., Som. Cell Mol. Genet., 16: 555-566 (1986)]. The medium was named CHO medium plus HT.

Five electrophoresis of 4 x 10 ⁶ cells and ⁵ ug of plasmid DNA [anti-CD4 (4 (PE) in NEPOSLA3F, Lambda, OZ) using a BTX 600 electrophoresis device (BTX, San Diego, Calif.) In a 0.4-pegable kvet -). Prior to electrophoresis, the plasmid was limited to Pac I, which isolates the gene expressed in mammalian cells, and part of the plasmid was used to grow the plasmid in bacteria. The conditions for electrophoresis are 230V, 400 F and 13. Each electrophoresis was plated into one 96-well dish (about 40,000 cells / well). After 2 days of electrophoresis and if necessary, the dish was fed with CHO medium plus HT (Geneticin, GIBCO) containing G418 at 400 ug / active compound until colonies occurred. Supernatants from confluent colonies were analyzed for the presence of chimeric immunoglobulins by ELISA specific for human antibodies. Twenty eight G418 resistant colonies occurred in five plates (from 480 wells). Most of the antibodies, clones, G418 resistant colonies expressing 5Cl were confluent after 30 days of electrophoresis. Southern blot analysis showed that clone 5Cl was a single replication component (not shown). In a 4-day culture sprayed at 10 ⁵ cells / at 125 spinnerets, the clones doubled every 28 hours and the antibody production rate was 0.5 pg / cell / day (0.9 / L).

Amplification

Clone 5Cl was weighed and plated into 96-well dishes (MTX, Sigma ( ^* ) amethoferin) containing CHO medium + 5 nM methotrexate at various concentrations from 10 ⁶ cells / plate to 3 × 10 ⁴ cells / plate I was. After 20 days, clone 5Cl-5B9 formed confluence on 3 × 10 ⁵ cells / plate (49 of 96-wells grew on the plates). The clones were weighed. In 4 day cultures seeded at 10 ⁵ cells / at T150, the clones doubled every 35.5 hours and antibody reduction was 15.3 pg / cell / day (18 / L). Clone 5Cl-5B9 was weighed and plated into 96-well dishes containing CHO medium + 5 nM methotrexate at various concentrations from 100 cells / plate to 3 × 10 ⁴ cells / plate. After 36 days, clone 5Cl-5B9 50Cl formed about 60% confluence on 10 ⁵ cells / plate (50 of 96-wells grew on the plate). The clones were weighed.

Cell Bank for Phase I Supply of CE94PE Mother Seed Stock Solution (PSS)

50 nM MTX PSS of clone 5Cl-5B9-50Cl was frozen. Cells were cultured in 500 spinners containing CHO and 50 nM MTX. Upon freezing, the cultures reached a density of 1.1 × 10 ⁶ cells /, with 96% variation and 29.3 hours doubling time. Antibody production was determined to be about 27 pg / cell / day by sandwich ELISA. Cells were centrifuged from the medium and placed in a glass bottle at a density of 2.0 × 10 ⁷ cells / in 95% JRH Bioscience Bovine Serum and 5% Sigma (Common Master Mixture), and freezing medium 1 with cells in a glass bottle. Frozen. The vial was frozen at -70 and placed in a liquid nitrogen tank the next day.

One 50 nM MTX PSS vial was thawed and seeded in 100 ml spinners containing CHO medium and 50 nM MTX. After 3 days, the spinner was separated from 2 × 10 ⁵ cells / ml into 2 × 125 ml spinners; One spinner containing CHO medium and 50 nM MTX and another spinner containing only CHO medium.

After 3 days, only 15 ml of cells and medium from Spiter's CHO medium were frozen and the points for the Consider Mycoplasma test were sent to Tektagen. Both production runs were performed for 8 weeks with or without MTX. Results from tecgenogens include anti-CD4 (gamma 4 (PE), lambda, OZ-) NEOSPLA3F in CHO; Mycoplasma-free clone 5C1-5B9-50C1 parent seed stock. Ten vials of 50 nM NM PSS were transferred for storage in liquid nitrogen.

Master Cell Bank (MCB)

Two 50 mM MTX PSS vials of clone 5C1-5B9-50C1 were thawed and seeded into 100 ml spinner flasks containing CHO medium and 50 nM MTX. Cultures were expanded for 6 days into a larger spinner flask until a volume of 2000 ml with a density of 9.5 × 10 ⁵ and a variability of 98% was obtained. Cells were centrifuged from the medium and resuspended at a density of 2.0 × 10 ⁷ cells / ml in 95% JRH Biosciences Fetal Bovin Serum and 5% Sigma Hybrimax DMXO. The cell suspension in freezing medium was divided (10 ml) into each 80 cryogenic vial designed as MCB G4PE50-MA. The vial was frozen at -70. After 24 hours, cell banks were transferred for storage in liquid nitrogen.

Example 5

Stability of CD4 mAb

The physical and chemical stability of CE94PE and CE94E solutions were monitored at 5, 40 over 3 months and lighted in SDS-PAGE (reduced and unreduced), IEF, reverse phase-HPLC, size exclusion chromatography (SEC) and ELISA Diffused. Initial testing by RP-HPLC and unreduced SDS-PAGE showed that CE94PE consisted of two main species, unreduced whole molecules and noncovalently bound molecules, which were labeled "halfmer" under assay conditions. To separate two identical units. Interestingly, no major differences in the bio-assay profiles of the two mAbs are observed by SEC, IEF, SDS-PAGE (reduced) and ELISA. The amount of "halmer" in CE94PE by RP-HPLC or SDS-PAGE (unreduced) is less than 1%. FIG. 18 shows SDS-PAGE (unreduced) of monomers and “halfmers” in solutions of CE94PE and CE94E. The amount of "halmer" in CE94E remains constant with respect to the initial test under any of the conditions tested over three months. The content of "halfmer" in CE94PE remained below 2% at all time conditions tested. No difference in stability of CE94E and CE94PE was observed at 5 and 40. CE94E stored under light diffusion is slightly less stable than CE94PE. The data suggest that the "halmer" in CE94E does not significantly affect the overall stability of the entire molecule. No major difference in physical stability of CE94PE and CE94E was observed.

Affinity and Stoichiometry of CD4 mAb by Surface Plasmon Resonance

The stoichiometry of soluble CD4 binding to immobilized mAbs can be measured by saturation binding experiments in BIAcore (Pharmacia). Data on the association and dissociation phases of the SPR process (FIG. 19) can be found in O'Shannessey et al., Anal. Biochem., 212: 467-468 (1993)] and analyzed directly using the integral form of the rate equation. A summary of binding data expressed in mole CD4 / mole mAb is shown in Table 3. From the data it can be seen that in all cases, the stoichiometry of the bond is greater than 1.5: 1. If BIAcore is a solid phase interaction system and the immobilization protocol is optional, the results suggest that both antigen binding regions of each mAb are functional. Thus, the stoichiometry of CD4 binding is the same for all mAbs and is close to a theoretical value of 2.0. In addition, affinity measurements show the same affinity for all mAb complexes, ie about 1.0 nM.

TABLE 3

Stoichiometry and Affinity of Bonds Measured by Surface Plasmon Resonance

Antibodies BIAcore stoichiometry Affinity (nM 25) CE9.1 1.56 0.99 CE94 1.61 1.34 CE94E 1.72 1.43 CE94K 1.67 1.08 CE94PE 1.09

Example 6

In Vitro Biological Evaluation of CE94PE

summary

CE9.1, CE94PE and other gamma 4 derivatives were compared for activity mediated by the Fab region (MLR) and Fc domains (Fc receptor binding, ADCC and CDC). Fab dependent activity (MLR) did not differ between mAbs, but their Fc receptor binding properties were different. Unmodified gamma 4 induced CE94 showed surprisingly strong binding to the Fc receptor, while E mutations in CE4E and CE94PE eliminated this binding and ADCC activity.

Effect of mAb on Mixed Lymphocyte Response (MLR)

Mixed lymphocyte response (MLR) assays were performed in three ways to determine the effect of mAb composition on all antigen induced T cell proliferation and EL-2 production. MLR is dependent on the CD4 ⁺ T cells of the invention and most responses are dependent on the participation of CD4 receptors through interaction with MHC Glass II molecules on antigens present in the cells. MLR responses represent an in vitro correlation with respect to transplant rejection in vivo. In terms of other drugs, the MLR is also blocked by immunosuppressive agents such as cyclosporin A.

All mAb constructs were equivalent in blocking MLR and were both read as T cell proliferative response and IL-2 production (FIG. 20 and Table 4). Thus, the “P & E” substitutions and CH2 domains in the V domain histograft and hinge of CE9.1 on human tetrastructure did not affect the ability of mAbs to block CD4 dependent T cell reactants in vitro.

Table 4

Effect of mAb on MLR-Summary

Antibodies Proliferation (IC50) IL-2 generation (IC50)  CE9.1 20 ng / 5 ng / CE94 20 ng / 5 ng / CE94E 20 ng / 10 ng / CE94K 20 ng / 20 ng / CE94PE 20 ng / ND

conclusion

All mAbs were equivalent in inhibition of MLR.

Fc Receptor Binding Properties of mAb

Assay confirmation for measurement of Fc receptor binding

CE94PE was designed to eliminate FcR binding activity. To measure this activity, assays were developed based on FcR and CD4-mediated adhesion of cells by mAbs via bridging. This assay simultaneously measured the CD4 and FcR binding function of mAb as an in vitro correlation to FcR and mAb mediated clearance of CD4 cells in vivo.

21 demonstrates that CE9.1 facilitates attachment of FcR expressing monocytes (IFN-induced THP-1 cells) to CD4 ⁺ fibroblasts (CD4 transfected fibroblast cell line) in an attachment assay. will be. Binding depends on the Fc domain of mAb CE9.1 since the F (ab ') 2 with the truncated end cannot facilitate the binding. Binding also requires an antigen recognition region of CE9.1 because its occupancy with sCD4 blocks cell-cell adhesion.

Measurement of Fc Receptor Binding Activity of mAb

mAbs CE9.1 (IgG1), CE94 (IgG4), CE94K (IgG4 K, hybrid), CE94E (IgG4, E mutant) and CE94PE (IgG4, PE mutant) have the ability to simultaneously bind cell surface CD4 and FcR. Was evaluated.

As expected, CE9.1 had good binding activity in this assay. Surprisingly, the IgG4 constructs, CE94 and CE94K, had sufficient affinity for FcR, indistinguishable from CE9.1 in this assay. Activity in this assay was only lost when the "E" substituent was introduced as in CE94E and CE94PE (see Figure 22).

In Vitro Clq Binding Properties of CE94PE

The complement system contains the ability to interact with specific types of antibodies in a way that causes cell lysis and destruction among several functional elements. Human IgG1 antibodies generally have the ability to bind Clq and eliminate target cells containing surface antigens with specificity. Other human isotypes, such as IgG4, show reduced ability to bind Clq, and thus will not be able to eliminate target cells. The manipulation of CE94PE in gamma 4 constructs could theoretically serve the purpose of inhibiting complement binding and allow the antibody to bind to CD4 target cells, which eliminates potentially damaging side effects.

The CDC effector properties of CE94PE and CE9.1 were compared using the classical method of complement mediated cytolysis of chromium labeled CD4 ⁺ SupT1 cells in the presence of rabbit complement. In these studies, murine complement binding mAbs to HuCD4, 4D9 (IgG2a) were used as positive controls. CE9.1 and CE94PE were both ineffective at binding earthenware complement (FIG. 23). It was earlier known that CE9.1 was incompletely bound to Clq and therefore could not bind human complement. These results show that both constructs cannot promote cell lysis through the complement effector mechanism.

In Vitro ADCC Effector Characteristics of CE94PE

Cells with potential cytotoxicity capable of binding mAb by FcR can mediate ADCC induced into antibody coated target cells. Human T helper cells expressing CD4 molecules are recognized by mAb CE9.1, which stimulates cytolytic attack by FcR containing killer cells, granulocytes, and / or macrophages. The objective in the manipulation of CE94PE is to eliminate the ability of the mAb to bind to FcR, which removes the ability of helper cells to mediate the removal of CD4 target cells while allowing the mAb to retain immunosuppressive properties.

Comparison of ADCC effector properties of CE94PE and CE9.1 was achieved using the classical method of cell mediated cytolysis of chromium labeled CD4 ⁺ SupT1 cells. Murine CD4 mAb 4D9 (IgG2a, K) was selected as a positive control. Effector cells were human peripheral blood leukocytes obtained from the smoke screen. 12 shows the ability of mAb 4D9 and CE9.1 to mediate specific lysis of CD4 ⁺ cells. Under the same conditions, CE94PE showed little effect.

These results show that CE94PE cannot mediate cell lysis through FcR or complement mechanism.

Example 7

Pharmacodynamic Comparative Analysis of CE94E and CE94PE

Pharmacokinetic comparisons of the two lead 4 mAbs, CE94E and CE94PE were investigated in male Sprague-Dawley rats. CE94E or CE94PE was administered at 1 / (4 per group) as an intravenous pill and blood samples were taken for 4 weeks after administration. Plasma CE94E and CE94PE concentrations were measured using an sCN4 / anti human IgG sandwich ELISA designed to confirm the presence of circulating human IgG as well as the ability to bind recombinant human soluble CD4.

After 1 / iv intravenous pill administration of CD4 mAB, CE94E plasma, the concentration was reduced by a three step method and the CE94PE plasma concentration was reduced by a two step method (FIG. 24). Due to the insufficient number of data points to adequately describe the completion status for CE94E and the purpose of the comparison, all plasma concentration-time profiles were analyzed using a two-step model. A slight change in the interior was observed. The predominant second stage t _1/2 was about 4 days for CE94E and 9 days for CE94PE, calculated as 67% and 93% of the total area under the plasma concentration-time curve, respectively. The apparent plasma removal rate of CE94PE was low (6.4 / hr /), and about a fraction of the removal rate of CE94E (1.0 / hr /). Thus, the pharmacodynamic characteristics of the PE mutant 4 mAb, CE94PE, are similar to other humanized 1 mAbs in mice.

The long circulatory half-life of CE94PE, which is not functionally impaired in mice, also suggests that CE94PE would be effective for extended periods of time when administered to humans.

These results confirm that the PE mutant CE94PE has twice the plasma clearance and long half-life than the CE9E mutant in mice.

Table 5

Pharmacodynamic parameters (mean ± SD) of CE94E and CE94PE after administration of 1 / pill to Sprague-Dawley rats

CD4 mAb CL (/ hr /) AUC (ug% hr /) MRT (hr) T _1/2 ^-1 (hr) T _1/2 ^-2 (hr) % AUC ₂ V _SS (/) CE94E 0.999 ± 37 1010 ± 30 109 ± 2 15.2 ± .8 97.7 ± 9.5 55.7 ± 4.2 109 ± 4 CE94E 1.32 ± .30 760 ± 38 79.5 ± 6.0 16.6 ± .9 95.3 ± 0.8 52.1 ± 0.0 103 ± 8 CE94PE 0.410 ± .074 2500 ± 40 295 ± 3 9.9 ± .3 224 ± 3 93.4 ± .2 119 ± 6 Abbreviation of pharmacological parameter: CL = total plasma clearance; AUC _0-inf = total area under plasma concentration over time curve; MRT = mean residence time; T _1/2 ^-2 = apparent half-life in the second stage; % AUC ₂ =% area under plasma concentration over time curve during the second step; V _SS = volume of distribution in the stationary phase

Based on the results, CE94PE should be suitable for treatment, for example by in vivo administration. In addition, other routes of administration may also be suitable.

Example 8

HuCD4 ⁺  In Vivo Pharmacodynamic Study of Transgenic Mice

Description of HuCD4 Transgenic Mice

Introduction

The high species specificity of CE91 and CE94PE makes it difficult to assess in vivo efficiency. Pharmacodynamic responses to CE9.1 can be readily observed in chimpanzees through dose dependent clearance of CD4 ⁺ cells. The absence of this activity expected in CE94PE led to the use of other means for assessing efficacy. In particular, efficacy is observed in HuCD4 transgenic mice. The research in this system is described below.

HuCD4 ⁺  Gene implants

In HuCD4 transgenic mice developed at UCSF (Killeen et al., EMBO J., 12: 1547-53 (1993)), endogenous MuCD4 genes were cleaved by homologous recombination, and human CD4 mini gene transplantation of the MuCD4 promoter Introduced under control. In these mice, HuCD4 replaces MuCD4 in these mice heterocrossed for homozygosity. HuCD4 restores positive and negative selection in the thymus, which leads to the introduction of a single positive peripheral CD4 ⁺ or CD8 ⁺ T cells in minutes which can be counted as those found in normal mice. In addition, mature HuCD4 T cells as compared to MuCD4 knockout mothers demonstrate similar characteristics to their normal murine counterparts: (1) in vivo, serum IgG levels are restored to normal levels, and (2) such animals They show a suitable MHC dependent response in an in vitro NMR reaction.

The genetic background of these mice is rather complicated due to the need to use different strains in embryonic hepatocytes and mice and early knockout and gene transfer experiments. Initial knockout / gene transplanted mice were then cross-bred for graft conjugation at the MHC position, and the mice in use in SB are H-2 ^dd haplotypes.

The results showed that a good response to the foreign antigen, egg albumin was obtained in these HuCD4 mice, and initial studies demonstrated in vivo activity against CE94PE.

Prospective Evaluation of CE94PE in HuCD4 Gene Transplantation Mice

Twelve HuCD4 transgenic mice (H-2 ^dd ) were received and used to compare CE94PE with IF3 (murine antihuman CD4) and GK1.5. Mice were intraperitoneally administered to mice at days -2, -1 and 0, and immunized with OVA 3 hours after the last dose. Mice were sacrificed after 2 weeks and serum and cells were evaluated for functional activity. OVA specific antibody response is shown in FIG. 25. As expected, mAbs against mouse CD4 (GK 1.5) had no effect on humoral responses, whereas two mAbs against HuCD4 blocked the response. CE94PE is more dramatic than two mAbs.

However, all mouse groups that responded to Con A and LS had slight changes in MLR and in response to egg albumin. This may be due to the fact that a group of animals includes both males and females, and of different ages.

MuCD4-/-(CD4 knockout) mice and HuCD4 + / + (gene transplanted mice) were treated with CE9.1, CE94PE or saline. This study was conducted for a 28 day period and samples were taken on days 1, 3, 7, 14 and 28. Three-color flow cytometry of splenocytes from these mice was used to sacrifice CD4 ⁺ and CD8 ⁺ T cells. The following antibodies were used to measure T cell levels: CD3-PE, OKT4-FITC, CD8-TC. The following antibodies were used to sacrifice T and B cells in these mice: CD3-PE, CD2-FITC, CD45-TC. To sacrifice CE9.1 or CE94PE coated cells, the following mAb panels were used: OKT4-PE, Leu3a-FITC, CD3-TC.

In both CE9.1 or CE94PE treated transgenic mice (HuCD4 + / +), all CD4 ⁺ cells were coated with antibodies on day 1. The coating remained for several days and was no longer detected on day 28. The total number of treated CD4 ⁺ cells in CE9.1 treated mice was significantly reduced even on day 28. In contrast, CE94PE treated mice did not reduce total CD4 ⁺ cells. Both antibodies showed evidence of CD4 receptor changes. Although the percentage of CD8 ⁺ cells increased mutually in CE9.1 treated mice, there is no evidence that these absolute levels are significantly affected by this treatment. Similarly, the number of CD8 ⁺ cells was not affected in CE94PE treated mice. In all experiments, for either antibody, the number of B cells was constant.

In vivo studies in chimpanzees

Six chimpanzees were injected with an increased dose of antibody CE94PE up to 15 /, followed by CD4 ⁺ Removal of T cells and / or changes in CD4 receptors from the cell surface were observed. Peripheral blood samples were taken from each champ and then 3 and 2 weeks later began to establish a baseline for CD4 T cells. In addition to CD4 ⁺ cells, CD3 ⁺ and CD8 ⁺ cell levels were also measured by flow cytometry. MAb OKT4, which binds to different portions of the CD4 molecule and does not compete for binding with CE94PE, was used as a control. By subtraction of CD8 ⁺ counts from total CD3 ⁺ count, there is a theoretical value for the number of CD4 ⁺ cells can be calculated. By comparing this value with CD4 ⁺ cells determined by obtaining OKT4, CD4 receptor changes can be distinguished from CD4 ⁺ cell clearance.

At the start of the study, each chimpanzee was injected intravenously with saline. Blood samples were taken immediately 3 and 14 days after injection. CE94PE (0.05 /) was injected into each chimp and blood samples were taken 3 and 14 days later. CD4 ⁺ cells were observed and provided with CE94PE at subsequent levels of dose if they were within the normal range. In all cases, each chimpanzee is in accordance with the following draft: brine, O.05 / CE94PE, 1.5 / CE94PE, saline, 15 / CE94PE.

After injection of saline or CE94PE at 0.05 /, there was no effect on CD4 levels. At 1.5 /, CD4 ⁺ cell coating was observed, with no CD4 ⁺ cell removal but transient and partial changes in the CD4 receptor from the cell surface. In CE94PE 15 /, CD4 ⁺ cell removal was not observed in either animal, but significant changes were observed in all animals. The change effect was temporary and returned to baseline after 14-21 days. No bad results were observed in either animal. CE94PE could be observed on the cell surface and in serum up to 2 days after administration.

CE94PE was designed to be a non-removable antibody, and even at relatively high doses of 15 /, no clearance was observed in all animals. CE94PE was stable in serum of chimpanzees and was present in circulating blood for up to 21 days.

Usage

Antibodies generated by the methods described above, or by equivalent techniques, can be purified by a combination of affinity and size exclusion chromatography to characterize functional biological assays. These assays include measuring specificity and binding affinity as well as effector function or complement binding associated with expressed isotypes such as ADCC. Such antibodies can be used as passive or active therapeutic agents for infectious diseases and transplants, including B4 lymphoma, numerous human diseases associated with CD4 expression and T cells, AIDS, autoimmune and inflammatory diseases. The antibody can be used in its native form or as part of an antibody / chelate, antibody / drug or antibody / toxo complex. In addition, the whole antibody or antibody fragments (Fab ₂ , Fab, Fv) can be used as imaging agents or as efficacious vaccines or immunogens in active immunotherapy against the development of antigenic responses.

The amount of antibody useful for obtaining a therapeutic effect can be determined by standard techniques well known to those skilled in the art. Antibodies will generally be provided by standard techniques within the pharmaceutically acceptable buffer range and can be administered by the preferred route. Because of the efficacy of the antibodies claimed herein and their resistance by humans, the administration of each of these antibodies can control several diseases or disease states in humans.

Anti-CD4 recombinant antibodies (or fragments thereof) of the invention are also useful for inducing immunomodulation, eg, for suppressing the immune system of a human or animal. Accordingly, the present invention relates to a method for inducing immunomodulation prophylactically or therapeutically in a human or other animal in need thereof by administering such an antibody of the present invention to a human or other animal in a nontoxic effective amount. .

The immunomodulatory inducibility of the compounds of the present invention can be demonstrated in standard tests used for this purpose, such as a mixed lymphocyte response test or a test measuring inhibition of T cell proliferation measured by thymidine uptake.

The fact that the antibodies of the invention are used to induce immunosuppressive methods suggests that treatment or prevention of resistance of organs or tissues (e.g., kidney, heart, lung, bone marrow, skin, cornea, etc.) into which the antibodies of the invention are implanted or denial; Treatment or prevention of autoimmune, inflammatory, proliferative and hyperproliferative diseases, and immunologically mediated diseases (eg, rheumatoid arthritis, lupus erythematosus, systemic lupus erythematosus, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, Type 1 Diabetes, Uveitis, Nephrotic Syndrome, Psoriasis, Atopic Dermatitis, Contact Dermatitis and other Eczema Dermatitis, Seborrheic Dermatitis, Squamous Gland, Pemphigus, Bullous Pelvic Blebs, Bullous Epidermolysis, Urticaria, Angioedema, Vasculitis, Treatment or prevention of skin signs of erythema, dermal eosinophilia, alopecia areata, etc .; Reversible obstructive airway disease, intestinal inflammation and allergies (eg, celiac disease, proctitis, eosinophilic gastroenteritis, mastocytosis, Crohn's disease and ulcerative colitis) and food-related allergies (eg, migraine, rhinitis and eczema) Means useful in the treatment of. In addition, the antibodies of the present invention are useful for treating non-immune conditions in which immunomodulation is desired, such as graft-versus-host disease (GVHD), transplant rejection, asthma, HIV, leukemia, lymphoma, and the like.

One skilled in the art will be able to determine the nontoxic effective amount of an antibody for immune suppression by routine experimentation. Generally, an effective dosage will be about 0.05 to 100 per body weight per day.

Antibodies (or fragments thereof) of the invention will also be useful for treating tumors in mammals. More specifically, the antibodies of the present invention will be useful for reducing tumor size, inhibiting tumor growth, and / or prolonging survival in tumored animals. Thus, the present invention also relates to a method of treating a tumor by administering a nontoxic effective amount of an antibody to such a human or animal. One skilled in the art will be able to determine the nontoxic effective amount of an antibody for treating a carcinogenic tumor by general experiments. In general, however, effective dosages are expected to be about 0.05-100 per body weight per day.

The antibodies of the invention can be administered to humans or other animals according to the method of treatment in an amount sufficient to produce a therapeutic or prophylactic effect. Such antibodies of the invention can be administered to humans or other animals in conventional dosage forms prepared by combining the antibodies of the invention with conventional pharmaceutically acceptable carriers or diluents according to known techniques. Those skilled in the art will appreciate that the form and character of the pharmaceutically acceptable carrier or diluent is determined by the amount of active ingredient to be combined, the route of administration and other well known parameters.

The route of administration of the antibodies (or fragments thereof) of the invention can be oral, parenteral, or topical by inhalation. As used herein, the term parenteral includes intravenous, intramuscular, subcutaneous, rectal, vaginal or intraperitoneal administration. Subcutaneous and intramuscular forms of parenteral administration are generally preferred.

Daily parenteral and oral dosages for the use of the compounds of the present invention to prophylactically or therapeutically induce immunosuppression or therapeutically treat carcinogenic tumors are generally from about 0.05 to 100 mg per body weight per day, preferably Will be about 0.5 to 10.

Antibodies of the invention can also be administered by inhalation. "Inhalation" means intranasal and oral inhalation administration. Suitable dosage forms for such administration, such as aerosol formulations or dose metering inhalers, can be prepared by conventional techniques. Preferred dosages of the compounds of the invention to be used are generally about 10 to 100.

Antibodies of the invention can also be administered topically. Topical administration means non-systemic administration, which involves the external application of an antibody (or fragment thereof) compound of the invention to the epidermis, buccal cavity, and injection of such antibody where there is no significant influx of ear, eye nose, and blood flow. It involves doing. Systemic administration means oral, intravenous, intraperitoneal and intramuscular administration. The amount of antibody required for a therapeutic or prophylactic effect will depend on the antibody selected, the nature and severity of the condition being treated, and the animal being treated, and ultimately determined by the physician. Suitable topical dosages of the antibodies of the invention will generally be about 1-100 per body weight per day.

Formulation

The antibody or fragment thereof may be administered alone, but it may be desirable to be in a pharmaceutical formulation. For topical administration, the active ingredient may comprise 0.001 to 10% (w / w) of the formulation, for example 1 to 2% by weight, but may comprise as much as 10% (w / w) of the formulation, 5 It is preferred not to exceed% (w / w), more preferably 0.1 to 1% (w / w).

Topical formulations of the invention include the active ingredient together with one or more acceptable carriers and optionally other therapeutic ingredients. The carrier should be acceptable in the sense of being compatible with the rest of the formulation and should not be harmful to its excipients.

Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration into the area in need of treatment through the skin, such as coagulants, lotions, creams, ointments or pastes.

Drops for the present invention may include sterile aqueous or oily solutions or suspensions and are active in suitable aqueous solutions of sterile and / or fungicides and / or other suitable preservatives (and preferably including surfactants). It can be prepared by dissolving the component. The resulting solution is then purified by filtration, transferred to a suitable container and sealed and sterilized by autoclaving or holding at 90-100 for 30 minutes. Optionally, the solution can be sterilized by filtration and transferred to the container by antiseptic technique. Examples of suitable fungicides and fungicides included in the drops include phenylmercury nitrate or acetate (0.002%), benzalkonium chloride (0.01%) and chlorhexidine acetate (0.01%). Suitable solvents for the preparation of oily solutions include glycerol, diluted alcohol and propylene glycol.

Lotions according to the present invention include those suitable for application to the skin or eyes. Lotions suitable for the eye may comprise a sterile aqueous solution, optionally including a fungicide, and may be prepared in a manner similar to those for the preparation of drops. Lotions or clogs for application to the skin may also include agents that promote dry skin and cool the skin, such as alcohol or acetone, and / or wetting agents, such as glycerol or castre oil or arachis oil. .

Creams, ointments or pastes according to the invention are semisolid formulations of the active ingredient for external application. They can be prepared by mixing the active ingredient in finely divided or powdered form, alone or in solution or with a fat or oil-free base, with the aid of a suitable machine as a suspension of an aqueous or non-aqueous fluid. The base may be selected from hydrocarbons such as hard, soft or liquid paraffin, glycerol, beeswax, metallic soaps; mucus; Natural oils such as almond, corn, arachis, castor or olibu oil; Wool or derivatives thereof, or fatty acids such as stearic acid or oleic acid and alcohols such as propylene glycol or macrogol. The formulations may incorporate suitable surfactants, such as anionic, cationic or nonionic surfactants, such as sorbitan esters or polyoxyethylene derivatives thereof. Other components such as natural gums, cellulose derivatives or silicatic silicas, and lanolin may also be included.

Those skilled in the art will determine that the optimal amount of an antibody or fragment thereof of the present invention and each administration interval is determined by the nature and extent of the condition being treated, the dosage form, the route and region, and the particular animal being treated, and such optimal amount may be determined by conventional techniques. It will be appreciated that it can be determined by. Those skilled in the art will also recognize that the optimal course of administration, the number of doses of the antibody or fragment of the invention given per day for a defined number of days, can be ascertained by those skilled in the art using the procedures of conventional therapeutic assays.

Without further adding, it is believed that one skilled in the art can, using the above description, utilize the present invention to its fullest extent. Accordingly, the following is to be construed as illustrative only, and in no way limit the scope of the invention.

Capsule composition

The pharmaceutical composition of the present invention in capsule form is prepared by filling a two-part hard gelatin capsule with the antibody of the present invention or fragment 50, lactose 100, talc 32 and magnesium stearate 8 in powder form.

Injectable Parenteral Composition

Pharmaceutical compositions of the present invention in a form suitable for administration by injection are prepared by stirring 1.5% by weight of the antibody or fragment thereof of the present invention in 10% by volume propylene glycol and water. This solution is sterilized by filtration.

Ointment composition

1.0 g of the antibody or fragment thereof of the present invention

White soft paraffin to reach 100.0 g

The antibody or fragment thereof of the invention is dispersed in a small amount of vehicle to produce a smooth homogeneous product. The dispersion is then filled in a metal extrusion tube.

Topical cream compositions

1.0 g of the antibody or fragment thereof of the present invention

Polarax GP 200 20.0g

2.0 g of lanolin anhydrous

2.5g white beeswax

0.1 g of methyl hydroxybenzoate

Distilled water to 100g

Paula wax, beeswax and lanolin are heated together to 60. Methyl hydroxybenzoate is added and homogenized by high speed stirring. The temperature is then lowered to 50. Thereafter, the antibodies or fragments of the invention are added, dispersed and the composition is cooled with slow stirring.

Topical Lotion Compositions

1.0 g of the antibody or fragment thereof of the present invention

0.6 g of sorbitan monolaurate

Polysorbate 20 0.6g

1.2 g of cetostearyl alcohol

Glycerin 6.0g

0.2 g of methyl hydroxybenzoate

Purified water B.P. (British Pharmacopeia) to 100.00

Methyl hydroxybenzoate and glycerin are dissolved in 75 to 70 water. Sorbitan monolaurate, polysorbate 20 and cetostearyl alcohol are dissolved together at 75 and added to an aqueous solution. The emulsion formed is homogenized and cooled with constant stirring and the antibody or fragment thereof of the invention is added as a suspension in the remaining water. Stir all of the suspension until homogeneous.

Eye drops composition

0.5 g of the antibody or fragment thereof of the present invention

0.01 g of methyl hydroxybenzoate

Propyl hydroxybenzoate 0.04 g

Purified water to 100.00 B.P.

Methyl and propyl hydroxybenzoate are dissolved in 75 to 70 purified water and the resulting solution is cooled. The antibody or fragment thereof of the invention is added and the solution is sterilized by filtration through a membrane filter (pore size 0.022) and filled into an antiseptic suitable sterilization container.

Compositions for Administration by Inhalation

For aerosol containers of 15 to 20 volumes: 10 antibodies of the invention or fragments thereof and 0.2 to 0.5% lubricant such as polysorbate 85 or oleic acid are mixed, and in a propellant such as freon, preferably (1,2 The mixture is dispersed in a combination of dichlorotetrafluoroethane) and difluorochloromethane and injected into a suitable aerosol container adapted for intranasal or oral inhalation administration.

Composition for administration by inhalation into a 15-20 dose aerosol container: Dissolving 10 antibodies of the invention or fragments thereof in ethanol (6-8) and a lubricant of 0.1-0.2%, such as polysorbate 85 or oleic acid Suitable aerosols which are added and dispersed in a propellant such as freon, preferably in a combination of (1,2 dichlorotetrafluoroethane) and difluorochloromethane and applied for intranasal or oral inhalation administration. Inject into the container.

The antibodies and pharmaceutical compositions according to the invention are particularly useful for parenteral administration, ie subcutaneous, intramuscular or intravenous administration. Compositions for parenteral administration will generally comprise a solution of the antibody or fragment thereof of the invention or a mixture thereof dissolved in an acceptable carrier, preferably an aqueous carrier. For example, various aqueous carriers can be used, such as water, buffered water, 0.4% saline, 0.3% glycine and the like. These solutions are sterile and generally contain no particulates. These solutions can be sterilized by conventional well known sterilization techniques. The composition may contain pharmaceutically acceptable auxiliary substances required for suitable physiological conditions such as pH adjustment and buffers and the like. The concentration of an antibody or fragment thereof of this invention in such pharmaceutical formulations can vary widely from less than about 0.5% by weight, generally from about 1% by weight or more, from 15% to 20% by weight, mainly depending on the particular dosage form selected, Will be selected based on viscosity and the like.

Thus, pharmaceutical compositions of the invention for intramuscular injection can be prepared to contain 1 sterile buffer water and 50 antibodies of the invention or fragments thereof. Similarly, pharmaceutical compositions of the invention for infusion into a vein can be prepared containing 250 sterile Ringer's solution, and 150 antibodies of the invention or fragments thereof. Substantial methods for preparing parenterally administrable compositions are known or described in more detail in literature, such as in Remington's Pharmaceutical Science , 15th ed., Mack Publishing Company, Easton, Pennsylvanina, and references herein. Cited by.

Antibodies (or fragments thereof) of the invention can be lyophilized for storage and can be reconstituted in a suitable carrier prior to use. The technique has been shown to be effective with conventional immunoglobulins, and known lyophilization and reconstitution techniques can be used.

Depending on the intended result, the pharmaceutical compositions of the present invention may be administered for prophylaxis and / or treatment. When applied therapeutically, the composition is administered to a patient already suffering from the disease in an amount sufficient to treat or at least partially arrest the disease and its complications. When applied prophylactically, a composition containing an antibody of the present invention or a mixture thereof is administered to a patient who is not yet in a disease state to increase patient tolerance.

Single or multiple administrations of the pharmaceutical compositions can be carried out at the level and form of dosage selected by the treating physician. In any case, the pharmaceutical composition of the present invention will provide a modified antibody (or fragment thereof) of the present invention in an amount sufficient to effectively treat the patient.

It is also noted that the antibodies of the present invention can be used for the construction and synthesis of peptides or non-peptide compounds (mimetics) that can be used for the same treatment as antibodies (Saragovi et al., Science , 253: 792-). 795 (1991)]

Deposit

Anti-CD4 in TCAE6 expressing strain XL1 blue, CE9.1 was deposited with ATCC, accession number 69030. The date of deposit is 9 July 1992.

Applicants and their assignees are obligated to replace such cultures five years or thirty years after the last request for cultures, or the deposit of such patent notices, before the period of the published patent is completed. It is meant to be informed and during this time deposits may be made available to the public unaltered. 37 C.F.R Sections 1-14 and 35 U.S.C. Section 112 can be obtained from the Commissioner of the Patent. Other aspects are within the scope of the following claims.

Sequence Listing

(1) General

(i) Applicants: Hannah, Nabil

Newman, Roland A.

Lev, Mitchell Lee.

(ii) Name of the Invention: Recombinant anti-CD4 antibody for use in human treatment

(Iii) number of sequences: 59

(Ⅳ) Contacts:

(A) Address: Burns, Money, Skipper & Matisse

(B) Street: 699 Princes Street

(C) City: Alexandria

(D) State: Virginia

(E) Country: United States

(F) Zip code: 22314-3187

(Iii) computer-readable form:

(A) Media type: floppy disk

(B) Computer: IBM PC compatible model

(C) working system: PC-DOS / MS-DOS

(D) Software: PatentIn Release # 1.0, Version # 1.30

(vi) Current application status:

(A) Application number: US 08 / 523,894

(B) Application date: 1995-9-6

(C) Classification:

(Ⅷ) Patent Attorney / Agent Representative:

(A) Statement: Teskin, Robin L.

(B) registration number: 35,030

(C) Reference / Document Number: 012712-165

(Iii) Communications:

(A) Telephone: (703) 836-6620

(B) Fax: (703) 836-2021

(2) Regarding SEQ ID NO: 1:

(i) the nature of the sequence:

(A) Length of sequence: 420 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) Origin:

(A) Creature: Monkey

(Iii) location in the dielectric:

(A) Chromosome / Short name: heavy chain variable domain of CE9.1

(Iii) the characteristics of the sequence:

(A) Name / Key: CDS

(B) Presence Location: 4..420

(Iii) the characteristics of the sequence:

(A) Name / key: mat_protein

(B) Presence Location: 61..420

(xi) Sequence description: SEQ ID NO: 1:

(2) Regarding SEQ ID NO 2:

(i) the nature of the sequence:

(A) Sequence length: 139 amino acids

(B) Sequence type: Amino acid

(D) mode: linear

(Ii) Type of sequence: protein

(xi) Sequence description: SEQ ID NO: 2:

(2) for SEQ ID NO 3:

(i) the nature of the sequence:

(A) Length of sequence: 387 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) Origin:

(A) Creature: Monkey

(Iii) location in the dielectric:

(A) Chromosome / Short name: light chain variable domain of CE9.1

(Iii) the characteristics of the sequence:

(A) Name / Key: CDS

(B) Presence Location: 4..387

(Iii) the characteristics of the sequence:

(A) Name / key: Matt _ protein

(B) Presence Location: 61..387

(xi) Sequence description: SEQ ID NO: 3:

(2) Regarding SEQ ID NO 4:

(i) the nature of the sequence:

(A) Length of sequence: 128 amino acids

(B) Sequence type: Amino acid

(D) mode: linear

(Ii) Type of sequence: protein

(xi) Sequence description: SEQ ID NO: 4:

(2) Regarding SEQ ID NO 5:

(i) the nature of the sequence:

(A) Length of sequence: 702 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) Origin:

(A) Biometric: Homo sapiens

(Iii) location in the dielectric:

(A) Chromosome / Short name: Lambda variable and constant domains at CE9.1

(Iii) the characteristics of the sequence:

(A) Name / Key: CDS

(B) Presence Location: 1..702

(Iii) the characteristics of the sequence:

(A) Name / key: Matt _ protein

(B) Presence Location: 1..702

(xi) Sequence description: SEQ ID NO: 5:

(2) Regarding SEQ ID NO 6:

(i) the nature of the sequence:

(A) Sequence length: 234 amino acids

(B) Sequence type: Amino acid

(D) mode: linear

(Ii) Type of sequence: protein

(xi) Sequence description: SEQ ID NO: 6:

(2) for SEQ ID NO 7:

(i) the nature of the sequence:

(A) Length of sequence: 1404 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) location in the dielectric:

(A) Chromosome / Short name: heavy chain variable and constant gamma 4

(Iii) the characteristics of the sequence:

(A) Name / Key: CDS

(B) Presence Location: 1..1404

(Iii) the characteristics of the sequence:

(A) Name / key: Matt _ protein

(B) Presence Location: 1..1404

(xi) Sequence description: SEQ ID NO: 7:

(2) Regarding SEQ ID NO: 8:

(i) the nature of the sequence:

(A) Length of sequence: 468 amino acids

(B) Sequence type: Amino acid

(D) mode: linear

(Ii) Type of sequence: protein

(xi) Sequence description: SEQ ID NO: 8:

(2) Regarding SEQ ID NO: 9:

(i) the nature of the sequence:

(A) Length of sequence: 1404 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) Origin:

(A) Biometric: Homo sapiens

(Iii) location in the dielectric:

(A) Chromosome / Short name: heavy chain gamma 4 with E mutation

(Iii) the characteristics of the sequence:

(A) Name / Key: CDS

(B) Presence Location: 1..1404

(Iii) the characteristics of the sequence:

(A) Name / key: Matt _ protein

(B) Presence Location: 1..1404

(xi) Sequence description: SEQ ID NO: 9:

(2) Regarding SEQ ID NO: 10:

(i) the nature of the sequence:

(A) Length of sequence: 468 amino acids

(B) Sequence type: Amino acid

(D) mode: linear

(Ii) Type of sequence: protein

(xi) Sequence description: SEQ ID NO: 10:

(2) Regarding SEQ ID NO 11:

(i) the nature of the sequence:

(A) Length of sequence: 1404 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) Origin:

(A) Biometric: Homo sapiens

(Iii) location in the dielectric:

(A) Chromosome / Short name: heavy chain gamma 4 with P and E mutations

(Iii) the characteristics of the sequence:

(A) Name / Key: CDS

(B) Presence Location: 1..1404

(Iii) the characteristics of the sequence:

(A) Name / key: Matt _ protein

(B) Presence Location: 1..1404

(xi) Sequence description: SEQ ID NO: 11.

(2) Regarding SEQ ID NO: 12:

(i) the nature of the sequence:

(A) Length of sequence: 468 amino acids

(B) Sequence type: Amino acid

(D) mode: linear

(Ii) Type of sequence: protein

(xi) sequence description: SEQ ID NO: 12:

(2) for SEQ ID NO: 13:

(i) the nature of the sequence:

(A) Sequence length: 26 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) Antisense: No

(Iii) Origin:

(A) biometric: human or monkey

(Iii) location in the dielectric:

(A) Chromosome / Short name: VH2 leader sequence

(xi) Sequence description: SEQ ID NO: 13:

(2) for SEQ ID NO: 14:

(i) the nature of the sequence:

(A) Length of sequence: 31 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) Antisense: No

(Iii) Origin:

(A) biometric: human or monkey

(Iii) location in the dielectric:

(A) Chromosome / Short name: VH2 leader sequence

(xi) Sequence description: SEQ ID NO: 14.

(2) Regarding SEQ ID NO: 15:

(i) the nature of the sequence:

(A) Sequence length: 29 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) Antisense: No

(Iii) Origin:

(A) biometric: human or monkey

(Iii) location in the dielectric:

(A) Chromosome / Short name: VH3 leader sequence

(xi) Sequence description: SEQ ID NO: 15:

(2) Regarding SEQ ID NO: 16:

(i) the nature of the sequence:

(A) Length of sequence: 31 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) Antisense: No

(Iii) Origin:

(A) biometric: human or monkey

(Iii) location in the dielectric:

(A) Chromosome / Short name: VH4 leader sequence

(xi) SEQ ID NO: 16:

(2) for SEQ ID NO: 17:

(i) the nature of the sequence:

(A) Length of sequence: 31 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) Antisense: No

(Iii) Origin:

(A) biometric: human or monkey

(Iii) location in the dielectric:

(A) Chromosome / Short name: VH5 leader sequence

(xi) SEQ ID NO: 17:

(2) Regarding SEQ ID NO: 18:

(i) the nature of the sequence:

(A) Length of sequence: 31 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) Antisense: No

(Iii) Origin:

(A) biometric: human or monkey

(Iii) location in the dielectric:

(A) Chromosome / Short name: VH6 leader sequence

(xi) Sequence description: SEQ ID NO: 18.

(2) Regarding SEQ ID NO: 19:

(i) the nature of the sequence:

(A) Sequence length: 30 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) Antisense: No

(Iii) Origin:

(A) biometric: human or monkey

(Iii) location in the dielectric:

(A) Chromosome / Short name: VH4 leader sequence with MulI region

(xi) Sequence description: SEQ ID NO: 19:

(2) Regarding SEQ ID NO: 20:

(i) the nature of the sequence:

(A) Sequence length: 30 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) Antisense: No

(Iii) Origin:

(A) biometric: human or monkey

(Iii) location in the dielectric:

(A) Chromosome / Short name: VH2 leader sequence with MluI region

(xi) Sequence description: SEQ ID NO: 20:

(2) Regarding SEQ ID NO: 21:

(i) the nature of the sequence:

(A) Sequence length: 27 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) Antisense: No

(Iii) Origin:

(A) biometric: human or monkey

(Iii) location in the dielectric:

(A) Chromosome / Short name: VH3 leader sequence with MulI region

(xi) Sequence description: SEQ ID NO: 21:

(2) Note to SEQ ID NO: 22:

(i) the nature of the sequence:

(A) Sequence length: 27 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) Antisense: No

(Iii) Origin:

(A) biometric: human or monkey

(Iii) location in the dielectric:

(A) Chromosome / Short name: VH4 leader sequence with MulI region

(xi) sequence description: SEQ ID NO: 22:

(2) Regarding SEQ ID NO: 23:

(i) the nature of the sequence:

(A) Sequence length: 30 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) Antisense: No

(Iii) Origin:

(A) biometric: human or monkey

(Iii) location in the dielectric:

(A) Chromosome / Short name: VH5 leader sequence with MulI region

(xi) Sequence description: SEQ ID NO: 23:

(2) Regarding SEQ ID NO: 24:

(i) the nature of the sequence:

(A) Sequence length: 23 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) Antisense: No

(Iii) Origin:

(A) biometric: human or monkey

(Iii) location in the dielectric:

(A) Chromosome / Short name: VH1, 3a, 5 primer with Xho I region

(xi) Sequence description: SEQ ID NO: 24:

(2) Regarding SEQ ID NO: 25:

(i) the nature of the sequence:

(A) Sequence length: 23 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) Antisense: No

(Iii) Origin:

(A) biometric: human or monkey

(Iii) location in the dielectric:

(A) Chromosome / Short name: VH2 primer with Xho I region

(xi) sequence description: SEQ ID NO: 25

(2) Regarding SEQ ID NO: 26:

(i) the nature of the sequence:

(A) Sequence length: 23 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) Antisense: No

(Iii) Origin:

(A) biometric: human or monkey

(Iii) location in the dielectric:

(A) Chromosome / Short name: VH3b primer with Xho I region

(xi) Sequence description: SEQ ID NO: 26:

(2) Regarding SEQ ID NO: 27:

(i) the nature of the sequence:

(A) Sequence length: 23 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) Antisense: No

(Iii) Origin:

(A) biometric: human or monkey

(Iii) location in the dielectric:

(A) Chromosome / Short name: VH4 primer with Xho I region

(xi) Sequence description: SEQ ID NO: 27:

(2) Regarding SEQ ID NO: 28:

(i) the nature of the sequence:

(A) Sequence length: 23 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) Antisense: No

(Iii) Origin:

(A) biometric: human or monkey

(Iii) location in the dielectric:

(A) Chromosome / Short name: VH6 primer with Xho I region

(xi) Sequence description: SEQ ID NO: 28:

(2) Regarding SEQ ID NO: 29:

(i) the nature of the sequence:

(A) Sequence length: 26 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) Antisense: No

(Iii) Origin:

(A) biometric: human or monkey

(Iii) location in the dielectric:

(A) Chromosome / Short name: IgG1-4 primer with NheI region

(xi) Sequence description: SEQ ID NO: 29:

(2) Regarding SEQ ID NO: 30:

(i) the nature of the sequence:

(A) Sequence length: 38 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) Antisense: No

(Iii) Origin:

(A) biometric: human or monkey

(Iii) location in the dielectric:

(A) Chromosome / Short name: kappa light chain primer with Bgl II region

(xi) SEQ ID NO: SEQ ID NO: 30:

(2) for SEQ ID NO: 31:

(i) the nature of the sequence:

(A) Length of sequence: 37 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) Antisense: No

(Iii) location in the dielectric:

(A) Chromosome / Short name: kappa light chain primer with Bgl II region

(xi) Sequence description: SEQ ID NO: 31:

(2) Regarding SEQ ID NO: 32:

(i) the nature of the sequence:

(A) Length of sequence: 41 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) Antisense: No

(Iii) Origin:

(A) biometric: human or monkey

(Iii) location in the dielectric:

(A) Chromosome / Short name: kappa light chain primer with Bgl II region

(xi) sequence description: SEQ ID NO: 32:

(2) Regarding SEQ ID NO: 33:

(i) the nature of the sequence:

(A) Length of sequence: 41 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) Antisense: No

(Iii) Origin:

(A) biometric: human or monkey

(Iii) location in the dielectric:

(A) Chromosome / Short name: kappa light chain primer with Bgl II region

(xi) sequence description: SEQ ID NO: 33:

(2) Regarding SEQ ID NO: 34:

(i) the nature of the sequence:

(A) Sequence length: 39 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) Antisense: No

(Iii) Origin:

(A) biometric: human or monkey

(Iii) location in the dielectric:

(A) Chromosome / Short name: Lambda light chain primer with Bgl II region

(xi) Sequence description: SEQ ID NO: 34:

(2) Regarding SEQ ID NO: 35:

(i) the nature of the sequence:

(A) Sequence length: 39 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) Antisense: No

(Iii) Origin:

(A) biometric: human or monkey

(Iii) location in the dielectric:

(A) Chromosome / Short name: Lambda light chain primer with Bgl II region

(xi) Sequence description: SEQ ID NO: 35:

(2) Regarding SEQ ID NO: 36:

(i) the nature of the sequence:

(A) Sequence length: 39 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) Antisense: No

(Iii) Origin:

(A) biometric: human or monkey

(Iii) location in the dielectric:

(A) Chromosome / Short name: Lambda light chain primer with Bgl II region

(xi) Sequence description: SEQ ID NO: 36:

(2) Regarding SEQ ID NO: 37:

(i) the nature of the sequence:

(A) Sequence length: 39 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) Antisense: No

(Iii) Origin:

(A) biometric: human or monkey

(Iii) location in the dielectric:

(A) Chromosome / Short name: Lambda light chain primer with Bgl II region

(xi) sequence description: SEQ ID NO: 37:

(2) Regarding SEQ ID NO: 38:

(i) the nature of the sequence:

(A) Sequence length: 38 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) Antisense: No

(Iii) Origin:

(A) biometric: human or monkey

(Iii) location in the dielectric:

(A) Chromosome / Short name: Lambda light chain primer with Bgl II region

(xi) Sequence description: SEQ ID NO: 38:

(2) Regarding SEQ ID NO: 39:

(i) the nature of the sequence:

(A) Length of sequence: 36 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) antisense: yes

(Iii) Origin:

(A) biometric: human or monkey

(Iii) location in the dielectric:

(A) Chromosome / Short name: kappa light chain primer with Kpn1 and BsiW1 regions

(xi) sequence description: SEQ ID NO: 39:

(2) Regarding SEQ ID NO: 40:

(i) the nature of the sequence:

(A) Sequence length: 30 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) antisense: yes

(Iii) Origin:

(A) biometric: human or monkey

(Iii) location in the dielectric:

(A) Chromosome / Short name: kappa light chain primer with Kpn1 and BsiW1 regions

(xi) sequence description: SEQ ID NO: 40:

(2) Regarding SEQ ID NO: 41:

(i) the nature of the sequence:

(A) Sequence length: 30 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) antisense: yes

(Iii) Origin:

(A) biometric: human or monkey

(Iii) location in the dielectric:

(A) Chromosome / Short name: kappa light chain primer with Hind III and Kpn1 regions

(xi) Sequence description: SEQ ID NO: 41:

(2) Regarding SEQ ID NO: 42:

(i) the nature of the sequence:

(A) Length of sequence: 36 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) antisense: yes

(Iii) Origin:

(A) biometric: human or monkey

(Iii) location in the dielectric:

(A) Chromosome / Short name: Lambda light chain primer with Kpn1 region

(xi) Sequence description: SEQ ID NO: 42:

(2) Regarding SEQ ID NO: 43:

(i) the nature of the sequence:

(A) Sequence length: 27 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) antisense: yes

(Iii) Origin:

(A) biometric: human or monkey

(Iii) location in the dielectric:

(A) Chromosome / Short name: lambda light chain primer having AvrII region

(xi) sequence description: SEQ ID NO: 43:

(2) Regarding SEQ ID NO: 44:

(i) the nature of the sequence:

(A) Sequence length: 17 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) Antisense: No

(Iii) Origin:

(A) biometric: human or monkey

(Iii) location in the dielectric:

(A) Chromosome / Short name: VH1 heavy chain variable region

(xi) sequence description: SEQ ID NO: 44:

(2) Regarding SEQ ID NO: 45:

(i) the nature of the sequence:

(A) Sequence length: 20 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) Antisense: No

(Iii) location in the dielectric:

(A) Chromosome / Short name: VH2 heavy chain variable region

(xi) Sequence description: SEQ ID NO: 45:

(2) Regarding SEQ ID NO: 46:

(i) the nature of the sequence:

(A) Sequence length: 20 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) Antisense: No

(Iii) location in the dielectric:

(A) Chromosome / Short name: VH3 heavy chain variable region

(xi) sequence description: SEQ ID NO: 46:

(2) Regarding SEQ ID NO: 47:

(i) the nature of the sequence:

(A) Sequence length: 20 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) Antisense: No

(Iii) location in the dielectric:

(A) Chromosome / Short name: VH4 heavy chain variable region

(xi) Sequence description: SEQ ID NO: 47:

(2) Regarding SEQ ID NO: 48:

(i) the nature of the sequence:

(A) Sequence length: 20 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) Antisense: No

(Iii) location in the dielectric:

(A) Chromosome / Short name: VH5 heavy chain variable region

(xi) Sequence description: SEQ ID NO: 48:

(2) Regarding SEQ ID NO: 49:

(i) the nature of the sequence:

(A) Sequence length: 20 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) Antisense: No

(Iii) location in the dielectric:

(A) Chromosome / Short name: VH6 heavy chain variable region

(xi) Sequence description: SEQ ID NO: 49:

(2) Regarding SEQ ID NO: 50:

(i) the nature of the sequence:

(A) Sequence length: 16 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) antisense: yes

(Iii) location in the dielectric:

(A) Chromosome / Short name: IgM heavy chain constant region

(xi) Sequence description: SEQ ID NO: 50:

(2) Regarding SEQ ID NO: 51:

(i) the nature of the sequence:

(A) Sequence length: 17 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) antisense: yes

(Iii) location in the dielectric:

(A) Chromosome / Short name: IgG1-4 heavy chain constant region

(xi) Sequence description: SEQ ID NO: 51:

(2) for SEQ ID NO: 52:

(i) the nature of the sequence:

(A) Sequence length: 21 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) location in the dielectric:

(A) Chromosome / Short name: kappa light chain variable region

(xi) sequence description: SEQ ID NO: 52:

(2) Regarding SEQ ID NO: 53:

(i) the nature of the sequence:

(A) Sequence length: 21 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) location in the dielectric:

(A) Chromosome / Short name: lambda light chain variable region

(xi) Sequence description: SEQ ID NO: 53:

(2) Regarding SEQ ID NO: 54:

(i) the nature of the sequence:

(A) Length of sequence: 19 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) antisense: yes

(Iii) location in the dielectric:

(A) Chromosome / Short name: kappa light chain constant region

(xi) sequence description: SEQ ID NO: 54:

(2) Regarding SEQ ID NO: 55:

(i) the nature of the sequence:

(A) Sequence length: 20 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) antisense: yes

(Iii) location in the dielectric:

(A) Chromosome / Short name: lambda light chain constant region

(xi) sequence description: SEQ ID NO: 55:

(2) Regarding SEQ ID NO: 56:

(i) the nature of the sequence:

(A) Sequence length: 30 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) location in the dielectric:

(A) Chromosome / Short name: PCR primers for human gamma 4 constant region

(xi) Sequence description: SEQ ID NO: 56:

(2) Regarding SEQ ID NO: 57:

(i) the nature of the sequence:

(A) Length of sequence: 31 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) location in the dielectric:

(A) Chromosome / Short name: PCR primers for human gamma 4 constant region

(xi) sequence description: SEQ ID NO: 57:

(2) Regarding SEQ ID NO: 58:

(i) the nature of the sequence:

(A) Sequence length: 96 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) location in the dielectric:

(A) Chromosome / Short Name: PCR Mutant of Human Gamma 4

(xi) sequence description: SEQ ID NO: 58:

(2) Regarding SEQ ID NO: 59:

(i) the nature of the sequence:

(A) Sequence length: 27 base pairs

(B) Type of sequence: nucleic acid

(C) Number of chains: 1 chain

(D) mode: linear

(Ii) Type of sequence: DNA (genome)

(Iii) location in the dielectric:

(A) Chromosome / Short Name: PCR Mutant of Human Gamma 4

(xi) SEQ ID NO: SEQ ID NO: 59:

Claims (42)

The heavy chain variable region polypeptide sequence of the Old World monkey monoclonal antibody shown in SEQ ID NO: 2 (FIG. 1), Light chain variable region polypeptide sequence of the Old World monkey monoclonal antibody shown in SEQ ID NO: 4 (FIG. 2), Human kappa or lambda light chain constant region, and Human gamma-4 (E) heavy chain constant region mutated by a change in leucine to glutamic acid at the position corresponding to residue 236 (residue 248, Kabat numbering) in the amino acid sequence shown in SEQ ID NO: 8 (FIG. 4); and Leucine is changed to glutamic acid at the position corresponding to residue 236 (residue 248, Kabat numbering) in the amino acid sequence shown in SEQ ID NO: 8 (FIG. 4), and at residue 229 (residue 241, Kabat numbering) A mutated human gamma-4 heavy chain constant region selected from the group consisting of mutated human gamma-4 (PE) heavy chain constant regions by altering serine to proline at the corresponding position and substantially deficient in T cell clearance activity , Chimeric antibodies that specifically bind to human CD4. The chimeric antibody of claim 1, wherein the light chain is a human lambda light chain comprising the polypeptide sequence shown in SEQ ID NO: 6 (FIG. 3). The chimeric antibody of claim 1, wherein the heavy chain is a mutated human gamma-4 (E) heavy chain comprising the polypeptide sequence shown in SEQ ID NO: 10 (FIG. 5). The chimeric antibody of claim 1, wherein the heavy chain is a mutated human gamma-4 (PE) heavy chain comprising the polypeptide sequence shown in SEQ ID NO: 12 (FIG. 6). 3. The chimeric antibody according to claim 2, wherein the heavy chain is a mutated human gamma-4 (E) heavy chain comprising the polypeptide sequence shown in SEQ ID NO: 10 (FIG. 5). 3. The chimeric antibody of claim 2, wherein the heavy chain is a mutated human gamma-4 (PE) heavy chain comprising the polypeptide sequence shown in SEQ ID NO: 12 (FIG. 6). Recombinant DNA encoding a chimeric antibody according to claim 1. Recombinant DNA encoding a chimeric antibody according to claim 2. Recombinant DNA encoding a chimeric antibody according to claim 3. Recombinant DNA encoding a chimeric antibody according to claim 4. A recombinant DNA encoding a chimeric antibody according to claim 6 and providing for expression of this chimeric antibody. A method of producing a chimeric antibody specific for CD4, comprising expressing the recombinant DNA of claim 7 in a recombinant host cell. A method of producing a chimeric antibody specific for CD4, comprising expressing the recombinant DNA of claim 8 in a recombinant host cell. A method of producing a chimeric antibody specific for CD4, comprising expressing the recombinant DNA of claim 9 in a recombinant host cell. A method of producing a chimeric antibody specific for CD4, comprising expressing the recombinant DNA of claim 10 in a recombinant host cell. A method of producing a chimeric antibody specific for CD4, comprising expressing the recombinant DNA of claim 11 in a recombinant host cell. Therapeutic or prophylactic of claim mediated by CD4 ⁺ T cells comprising a chimeric antibody according to claim 1 or an effective amount of CD4 ⁺ T cells suitable for treating or preventing the self-immune and chronic inflammatory diseases, disorders or conditions related to the composition. Therapeutic or prophylactic of claim mediated by CD4 ⁺ T cells comprising a chimeric antibody according to 2, wherein the effective amount or CD4 ⁺ T cells suitable for treating or preventing the self-immune and chronic inflammatory diseases, disorders or conditions related to the composition. Therapeutic or prophylactic of claim mediated by CD4 ⁺ T cells comprising a chimeric antibody according to 3, wherein the effective amount or CD4 ⁺ T cells suitable for treating or preventing the self-immune and chronic inflammatory diseases, disorders or conditions related to the composition. Therapeutic or prophylactic of claim mediated by CD4 ⁺ T cells comprising a chimeric antibody according to 4, wherein the effective amount or CD4 ⁺ T cells suitable for treating or preventing the self-immune and chronic inflammatory diseases, disorders or conditions related to the composition. 18. The method of claim 17, wherein the autoimmune or chronic inflammatory disease, disorder, or condition is selected from the group consisting of rheumatoid arthritis, inflammatory bowel disease, psoriasis, insulin dependent diabetes mellitus, systemic lupus erythematosus, cirrhosis and multiple sclerosis. Composition. 19. The method of claim 18, wherein the autoimmune or chronic inflammatory disease, disorder, or condition is selected from the group consisting of rheumatoid arthritis, inflammatory bowel disease, psoriasis, insulin dependent diabetes mellitus, systemic lupus erythematosus, cirrhosis and multiple sclerosis. Composition. 20. The method of claim 19, wherein the autoimmune or chronic inflammatory disease, disorder, or condition is selected from the group consisting of rheumatoid arthritis, inflammatory bowel disease, psoriasis, insulin dependent diabetes, systemic lupus erythematosus, cirrhosis and multiple sclerosis. Composition. The method of claim 20, wherein the autoimmune or chronic inflammatory disease, disorder, or condition is selected from the group consisting of rheumatoid arthritis, inflammatory bowel disease, psoriasis, insulin dependent diabetes mellitus, systemic lupus erythematosus, cirrhosis and multiple sclerosis. Composition. To treat or prevent a disease, disorder or condition selected from the group consisting of leukemia, lymphoma, graft-versus-host disease, asthma, transplant rejection and HIV infection comprising a therapeutically or prophylactically effective amount of a chimeric antibody according to claim 1 Suitable compositions. To treat or prevent a disease, disorder or condition selected from the group consisting of leukemia, lymphoma, graft-versus-host disease, asthma, transplant rejection and HIV infection comprising a therapeutically or prophylactically effective amount of a chimeric antibody according to claim 2 Suitable compositions. For treating or preventing a disease, disorder or condition selected from the group consisting of leukemia, lymphoma, graft-versus-host disease, asthma, transplant rejection and HIV infection comprising a therapeutically or prophylactically effective amount of a chimeric antibody according to claim 3 Suitable compositions. To treat or prevent a disease, disorder or condition selected from the group consisting of leukemia, lymphoma, graft-versus-host disease, asthma, transplant rejection and HIV infection comprising a therapeutically or prophylactically effective amount of a chimeric antibody according to claim 4 Suitable compositions. To treat or prevent a disease, disorder or condition selected from the group consisting of leukemia, lymphoma, graft-versus-host disease, asthma, graft rejection and HIV infection comprising a therapeutically or prophylactically effective amount of a chimeric antibody according to claim 6 Suitable compositions. Rheumatoid arthritis, psoriasis, insulin dependent diabetes mellitus, systemic lupus erythematosus, cirrhosis, multiple sclerosis, Hashimoto thyroiditis, type 1 myxedema, thyroid addiction, comprising a therapeutically or prophylactically effective amount of the chimeric antibody according to claim 1 / Graves' disease, pernicious anemia, autoimmune atrophic gastritis, autoimmune heartitis, Addison's disease, premature menopause, Goodpasture syndrome, myasthenia gravis, male infertility, vulgaris, vulgaris, sympathetic ophthalmitis, pacoogenic ) Uveitis, nephrotic syndrome, atopic dermatitis, contact dermatitis, seborrheic dermatitis, eczema dermatitis, squamous swelling, pemphigus, blistering bleb, bullous epidermis, urticaria, angioedema, vasculitis, erythema, dermatitis eosinophilia, alopecia areata , Reversible obstructive airway disease, migraine, eczema, rhinitis, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, idiopathic leukopenia (leucopenia), primary biliary cirrhosis, active chronic hepatitis (HBs Ag negative), latent cirrhosis, Sjogren's syndrome, dermatitis, skin sclerosis, mixed connective tissue disease, discus lupus erythematosus, systemic vasculitis, celiac disease , proctitis, eosinophilic upper small intestine salt, mastocytosis, Crohn's disease or ulcerative colitis mediated by selected CD4 ⁺ T cells from the group consisting of or CD4 ⁺ diseases related to T cell, a suitable composition for treating or preventing a disorder or condition. Rheumatoid arthritis, psoriasis, insulin dependent diabetes mellitus, systemic lupus erythematosus, cirrhosis, multiple sclerosis, Hashimoto thyroiditis, type 1 myxedema, thyroid addiction, comprising a therapeutically or prophylactically effective amount of the chimeric antibody according to claim 2 / Graves' disease, pernicious anemia, autoimmune atrophic gastritis, autoimmune heartitis, Addison's disease, premature menopause, Goodpasture syndrome, myasthenia gravis, male infertility, vulgaris, vulgaris, sympathetic ophthalmitis, pacoogenic ) Uveitis, nephrotic syndrome, atopic dermatitis, contact dermatitis, seborrheic dermatitis, eczema dermatitis, squamous swelling, pemphigus, blistering bleb, bullous epidermis, urticaria, angioedema, vasculitis, erythema, dermatitis eosinophilia, alopecia areata , Reversible obstructive airway disease, migraine, eczema, rhinitis, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, idiopathic leukopenia (leucopenia), primary biliary cirrhosis, active chronic hepatitis (HBs Ag negative), latent cirrhosis, Sjogren's syndrome, dermatitis, skin sclerosis, mixed connective tissue disease, discus lupus erythematosus, systemic vasculitis, celiac disease , proctitis, eosinophilic upper small intestine salt, mastocytosis, Crohn's disease or ulcerative colitis mediated by selected CD4 ⁺ T cells from the group consisting of or CD4 ⁺ diseases related to T cell, a suitable composition for treating or preventing a disorder or condition. Rheumatoid arthritis, psoriasis, insulin dependent diabetes mellitus, systemic lupus erythematosus, cirrhosis, multiple sclerosis, Hashimoto's thyroiditis, type 1 myxedema, thyroid addiction, comprising a therapeutically or prophylactically effective amount of a chimeric antibody according to claim 3 / Graves' disease, pernicious anemia, autoimmune atrophic gastritis, autoimmune heartitis, Addison's disease, premature menopause, Goodpasture syndrome, myasthenia gravis, male infertility, vulgaris, vulgaris, sympathetic ophthalmitis, pacoogenic ) Uveitis, nephrotic syndrome, atopic dermatitis, contact dermatitis, seborrheic dermatitis, eczema dermatitis, squamous swelling, pemphigus, blistering bleb, bullous epidermis, urticaria, angioedema, vasculitis, erythema, dermatitis eosinophilia, alopecia areata , Reversible obstructive airway disease, migraine, eczema, rhinitis, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, idiopathic leukopenia (leucopenia), primary biliary cirrhosis, active chronic hepatitis (HBs Ag negative), latent cirrhosis, Sjogren's syndrome, dermatitis, skin sclerosis, mixed connective tissue disease, discus lupus erythematosus, systemic vasculitis, celiac disease , proctitis, eosinophilic upper small intestine salt, mastocytosis, Crohn's disease or ulcerative colitis mediated by selected CD4 ⁺ T cells from the group consisting of or CD4 ⁺ diseases related to T cell, a suitable composition for treating or preventing a disorder or condition. Rheumatoid arthritis, psoriasis, insulin dependent diabetes mellitus, systemic lupus erythematosus, cirrhosis, multiple sclerosis, Hashimoto thyroiditis, type 1 myxedema, thyroid addiction, comprising a therapeutically or prophylactically effective amount of the chimeric antibody according to claim 4 / Graves' disease, pernicious anemia, autoimmune atrophic gastritis, autoimmune heartitis, Addison's disease, premature menopause, Goodpasture syndrome, myasthenia gravis, male infertility, vulgaris, vulgaris, sympathetic ophthalmitis, pacoogenic ) Uveitis, nephrotic syndrome, atopic dermatitis, contact dermatitis, seborrheic dermatitis, eczema dermatitis, squamous swelling, pemphigus, blistering bleb, bullous epidermis, urticaria, angioedema, vasculitis, erythema, dermatitis eosinophilia, alopecia areata , Reversible obstructive airway disease, migraine, eczema, rhinitis, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, idiopathic leukopenia (leucopenia), primary biliary cirrhosis, active chronic hepatitis (HBs Ag negative), latent cirrhosis, Sjogren's syndrome, dermatitis, skin sclerosis, mixed connective tissue disease, discus lupus erythematosus, systemic vasculitis, celiac disease , proctitis, eosinophilic upper small intestine salt, mastocytosis, Crohn's disease or ulcerative colitis mediated by selected CD4 ⁺ T cells from the group consisting of or CD4 ⁺ diseases related to T cell, a suitable composition for treating or preventing a disorder or condition. Rheumatoid arthritis, psoriasis, insulin dependent diabetes mellitus, systemic lupus erythematosus, cirrhosis, multiple sclerosis, Hashimoto thyroiditis, type 1 myxedema, thyroid addiction, comprising a therapeutically or prophylactically effective amount of a chimeric antibody according to claim 6 / Graves' disease, pernicious anemia, autoimmune atrophic gastritis, autoimmune heartitis, Addison's disease, premature menopause, Goodpasture syndrome, myasthenia gravis, male infertility, vulgaris, vulgaris, sympathetic ophthalmitis, pacoogenic ) Uveitis, nephrotic syndrome, atopic dermatitis, contact dermatitis, seborrheic dermatitis, eczema dermatitis, squamous swelling, pemphigus, blistering bleb, bullous epidermis, urticaria, angioedema, vasculitis, erythema, dermatitis eosinophilia, alopecia areata , Reversible obstructive airway disease, migraine, eczema, rhinitis, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, idiopathic leukopenia (leucopenia), primary biliary cirrhosis, active chronic hepatitis (HBs Ag negative), latent cirrhosis, Sjogren's syndrome, dermatitis, skin sclerosis, mixed connective tissue disease, discus lupus erythematosus, systemic vasculitis, celiac disease , proctitis, eosinophilic upper small intestine salt, mastocytosis, Crohn's disease or ulcerative colitis mediated by selected CD4 ⁺ T cells from the group consisting of or CD4 ⁺ diseases related to T cell, a suitable composition for treating or preventing a disorder or condition. A recombinant DNA encoding a chimeric antibody according to claim 5 and providing for expression of this chimeric antibody. A method of producing a chimeric antibody specific for CD4, comprising expressing the recombinant DNA of claim 35 in a recombinant host cell. Therapeutic or prophylactic according to claim 5, wherein the effective amount mediated by CD4 ⁺ T cells comprising a chimeric antibody or CD4 ⁺ T cells, autoimmune or chronic inflammatory disease, a composition suitable for treating or preventing a disorder or disease related to, 38. The composition of claim 37, wherein the autoimmune or chronic inflammatory disease, disorder or condition is selected from the group consisting of rheumatoid arthritis, inflammatory bowel disease, psoriasis, insulin dependent diabetes mellitus, systemic lupus erythematosus, cirrhosis and multiple sclerosis. . Rheumatoid arthritis, psoriasis, insulin dependent diabetes mellitus, systemic lupus erythematosus, cirrhosis, multiple sclerosis, Hashimoto thyroiditis, type 1 myxedema, thyroid poisoning, comprising a therapeutically or prophylactically effective amount of the chimeric antibody according to claim 5 / Graves' disease, pernicious anemia, autoimmune atrophic gastritis, autoimmune heartitis, Addison's disease, premature menopause, Goodpasture syndrome, myasthenia gravis, male infertility, vulgaris, vulgaris, sympathetic ophthalmitis, pacoogenic ) Uveitis, nephrotic syndrome, atopic dermatitis, contact dermatitis, seborrheic dermatitis, eczema dermatitis, squamous swelling, pemphigus, blistering bleb, bullous epidermis, urticaria, angioedema, vasculitis, erythema, dermatitis eosinophilia, alopecia areata , Reversible obstructive airway disease, migraine, eczema, rhinitis, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, idiopathic leukopenia (leucopenia), primary biliary cirrhosis, active chronic hepatitis (HBs Ag negative), latent cirrhosis, Sjogren's syndrome, dermatitis, skin sclerosis, mixed connective tissue disease, discus lupus erythematosus, systemic vasculitis, celiac disease , proctitis, eosinophilic upper small intestine salt, mastocytosis, Crohn's disease or ulcerative colitis mediated by selected CD4 ⁺ T cells from the group consisting of or CD4 ⁺ diseases related to T cell, a suitable composition for treating or preventing a disorder or condition. A composition suitable for treating or preventing a disease selected from the group consisting of leukemia, lymphoma, graft-versus-host disease, asthma, transplant rejection and HIV infection comprising a therapeutically or prophylactically effective amount of a chimeric antibody according to claim 5. Therapeutic or prophylactic of claim 6 wherein mediated by CD4 ⁺ T cells comprising a chimeric antibody according to the effective amount or CD4 ⁺ T cells, autoimmune or chronic inflammatory disease, a composition suitable for treating or preventing a disorder or disease related to, 42. The composition of claim 41, wherein the autoimmune or chronic inflammatory disease, disorder or condition is selected from the group consisting of rheumatoid arthritis, inflammatory bowel disease, psoriasis, insulin dependent diabetes mellitus, systemic lupus erythematosus, cirrhosis and multiple sclerosis. .